DE69837975T2 - Diagnostic imaging method - Google Patents

Abstract

The invention provides a method of generating an image of an animate human or non-human animal subject previously administered with a contrast agent involving generating an image of at least a part of said subject to which said contrast agent has distributed, characterised in that said contrast agent is a composition of matter of formula I V - L - R where V is a vector moiety having affinity for an angiogenesis-related endothelial cell receptor, L is a linker moiety or a bond and R is a detectable moiety, characterised in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species providing a multiplicity of labels detectable in in vivo imaging.

Description

These This invention relates to diagnostic imaging techniques, with which a disease state using a target contrast agent can be displayed. In particular, the invention relates to the use of such contrast agents in which the targeting vector binds to receptors associated with angiogenesis. such Contrast agents can therefore for the diagnosis of, for example, malignant diseases, heart disease, inflammation-related Diseases, rheumatoid arthritis and Kaposi's sarcoma become.

New blood vessels can be formed by two different mechanisms:
Vasculogenesis or angiogenesis. Angiogenesis is the formation of new blood vessels by diversion of existing vessels. The primary stimulus to this process may be inadequate supply of cells in a tissue containing nutrients and oxygen (hypoxia). The cells can respond by secreting angiogenic factors, of which there are many; One example that is frequently referred to is vascular endothelial growth factor (VEGF). These factors initiate the secretion of proteolytic enzymes that break down the proteins of the basement membrane, as well as inhibitors that limit the action of these potentially harmful enzymes. The other prominent effect of angiogenic factors is to cause endothelial cells to migrate and divide. Endothelial cells bound to the basement membrane, which forms a continuous piece around blood vessels on the contraluminal side, do not undergo mitosis. The combined effect of the loss of binding and the signals from the angiogenic factor receptors is that the endothelial cells are caused to move, multiply, and rearrange themselves, and eventually synthesize a basement membrane around the new vessels.

angiogenesis is outstanding in the growth and remodeling of tissues, including the Wound healing and inflammatory Processes. Tumors need Initiate angiogenesis when they reach a millimeter size, to maintain their growth rate. Because angiogenesis of characteristic changes is accompanied in the endothelial cells and their environment is this process is a promising target for therapeutic intervention. The inhibition of angiogenesis is also considered to be a promising one Strategy for Considered an anti-tumor therapy. The transformations, the angiogenesis are also very promising for the diagnosis, an obvious one Example is the malignant disease, but the concept is justified too big Hopes of inflammation and a variety of inflammatory-related diseases, including Atherosclerosis, wherein the macrophages of early atherosclerotic lesions are possible sources of angiogenic factors. These factors can also affect the revascularization of parts of the myocardium affected by an infarct be what happens if a stenosis is resolved within a short time.

at angiogenesis are receptors involved in endothelial Cells are unique. The surface These cells are remodeled in preparation for the migration and hidden structures are exposed where the basement membrane is mined, in addition to the variety of proteins involved in creating and controlling proteolysis are involved.

• Anmerkung: Viele Hormone, Wachstumsfaktoren und andere Verbindungen, die an Zelloberflächenrezeptoren binden, können als Vektoren wirken durch Binden an ihre Rezeptoren, oder, wenn sie bereits an die Zelloberfläche gebunden sind, sind sie Targets für Vektoren, die an sie binden, z.B. Antikörper.

A number of known receptors / targets associated with angiogenesis are listed below in Table 1. In the case of tumors, the resulting network of blood vessels is usually disorganized with the formation of sharp kinks and arteriovenous shunts. Using the targeting principles described in the present disclosure, angiogenesis can be detected by the majority of imaging modalities used in the medical field. Table 1 Receptors / targets associated with angiogenesis Receptors / Targets α ₂ -Antiplasmin Basal membrane components basic Fibroblast Growth Factor (bFGF) Biglycan (dermatan sulfate proteglycan) Cartilage-derived inhibitor [inhibitor] CD34 collagen type I, IV, VI, VIII Decorin (dermatan sulfate proteoglycan) dermatan sulfate proteoglycans Endoglin endosialin endothelin epidermal growth factor (heparin-binding ) Fibrin Fibrinopeptide B Fibroblast Growth Factor, FGF-3, basic Fibronectin Flt-1 / KDR, Flt-4 (VEGF receptor) FLT-1 (fms-like tyrosine kinase) (VEGF-A receptor) Heparan sulfate Hepatocyte growth factor Hepatocyte Growth factor receptor (c-met) hyaluronan insulin-like growth factor insulin-like growth factor / mannose-6-phosphate receptor Integrins: β ₃ and β ₅ , integrin α _v β ₃ , integrin α ₆ β ₁ (laminin receptor), integrin α ₆ , inregrin β ₁ , integrin α ₂ β ₁ , integrin α ₅ (subunit of the fibronectin receptor), integrin α _v β ₅ , Fibrin Receptors Interferon-α, -β [Inhibitors] Interleukins: IL-8, IL-12 [Inhibitors] Jagged Gene Product Laminin Laminin Fragments Leukemia Inhibiting Factor Ly-6 (a lymphocyte activating protein) Matrix Metalloprotease-2 Metalloproteinases Metalloproteinase Inhibitors MHC Class II Notch Gene Product Placental Growth Factor Placental proliferin-related protein Plasminogen Plasminogen activator Plasminogen activator inhibitor-1 Plasminogen activator receptor Platelet-derived growth factor (eg BB type) Platelet-derived endothelial cell growth factor Platelet factor 4 [inhibitor] Pleiotropin Proliferin, proliferin-related protein Receptor tyrosine kinases Selectins: E-selectin SPARC Stress Proteins (Molecular Charperone) (Glucose-regulated, Heat Shock Families) Syndeca n Tissue inhibitor of metalloproteinases (eg TIMP-2) Thrombin Thrombin receptor activating tetradecapeptide Thrombospondin [inhibitor] TIE receptors (tyrosine kinases with Ig and EGF-like domains) Tissue factor transforming growth factor-α, β tumor growth factor-α tumor necrosis factor urokinase-like plasminogen activator receptor vascular endothelial Growth factor-A on vascular endothelial growth factor-related protein vascular endothelial growth factor A receptor Vitronectin von Willebrand factor

• Note: Many hormones, growth factors and other compounds that bind to cell surface receptors can act as vectors by binding to their receptors or, if already attached to the cell surface, are targets for vectors that bind to them, eg antibodies.

As discussed above, many undesirable conditions are associated with neovascularization or angiogenesis, the development or proliferation of new blood vessels. Examples of such conditions are listed below in Table 2. Table 2 - Diseases and indications associated with angiogenesis Diseases / Indications arteriovenous malformations astrocytomas atherosclerosis breast cancers choriocarcinomas colorectal cancers gingivitis glioblastomas gliomas hemangiomas (childhood, capillary) hepatomas hyperplastic endometrium inflammation (eg chronic) ischemic myocardium Kaposi's sarcoma lung cancers macular degeneration melanoma metastases neuroblastomas occluding peripheral artery disease osteoarthritis ovarian cancers pancreatic cancers prostate cancers psoriasis retinopathy (diabetic, proliferative) Rheumatoid arthritis Scleroderma Seminoma types Skin cancers Formation of a solid tumor Ulcerative colitis

The surface cells, endothelial cells, of such new blood vessels have concentrations of various surface or transmembrane receptors that are larger than normal, such as receptor tyrosine kinases (RTK), and it has been proposed to use radiolabelled or chromophore-labeled antibodies to such receptors Similarly, analogs of natural protein ligands labeled for such receptors, as a means of detecting the centers of angiogenesis (see, eg WO95 / 26364 (Orion), WO96 / 30046 (Genentech) and WO95 / 1 2414 (Repligen)).

peptidic However, ligands have relatively few binding sites for detectable Markers (reporters) and the binding of reporters in many places on such peptidic ligands will affect the conformations, which the ligand can accept. Another problem with peptides is that they are often unstable in vivo.

It Therefore, there is still a need for methods that produce images, in which are effective targeted contrast media with affinities for angiogenesis Associated receptors are used.

Therefore, the invention, in one aspect, provides a method of producing an image of a living human or non-human animal subject to which a contrast agent has previously been administered, comprising generating an image of at least a portion of the subject, characterized in that the Contrast agent is a composition of a material of formula (I), VLR (I) wherein V is a vector moiety having an affinity for an angiogenesis-related endothelial cell receptor, L is a linker moiety or a bond and R is a detectable moiety, characterized in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species, which provides a variety of markers detectable in in vivo imaging.

Where R is a macromolecular or particulate species having a Providing variety of markers can be those markers that individually detectable (e.g., paramagnetic or radioactive Species) or they can Interacting to produce a detectable material, e.g. by means of a cooperative magnetic phenomenon. Examples of such Multireporter include polychelates and polyionic species and ferromagnetic, ferrimagnetic and superparamagnetic particles.

In many cases the substance composition of the formula I becomes a characterizable Be connected. In others, it can be a combination of compounds be that bound or otherwise associated with each other, e.g. are conjugated. For the sake of simplicity, the composition of matter hereinafter referred to as a means.

Contrast agents comprising gas-containing reporters are described in our co-pending International Patent Application no. PCT / GB97 / 02958 , filed on October 28, 1997.

In In one aspect, the invention provides a method of production a picture of a living human or non-human (preferably mammal or bird) animal subject previously administered a contrast agent had been, e.g. into the vascular System or the Gi tract, and to create an image at least a part of the subject to whom the contrast agent is administered was, e.g. by X-radiation, MR, Ultrasound, Scintigraphy, PET, SPECT, Electrical Impedance, light or magnetometric imaging modalities, thereby in that, as the contrast agent, an agent of the formula I is used.

Viewed from another aspect, the invention provides a method of monitoring the effect of treating a human or non-human animal subject with a drug to combat an angiogenesis-associated condition, eg, a cytotoxic agent, the method comprising detecting the uptake a previously administered composition of the formula I. VLR (I) wherein V is a vector moiety having an affinity for an angiogenesis-related endothelial cell receptor, L is a linker moiety or a bond, and is a detectable moiety, characterized in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species providing a variety of markers detectable in vivo imaging by angiogenesis-related endothelial cell receptors by generating an image of at least a portion of the subject, wherein administration and detection are optional but preferred repeatedly, eg before, during or after treatment with the drug.

The Agents of formula I have three characteristic components: one Vector (V); a linker (L); and a reporter (R). The vector must have the ability have the connection in one area of angiogenesis to the target To bring the reporter into an in vivo diagnostic imaging procedure be detectable; and the linker needs the vector to the reporter couple, at least until the reporter to the field of angiogenesis and preferably until the image-forming procedure is completed is.

vectors

When the vector may be any peptide or more preferably non-peptidic Connection with an affinity for receptors, associated with angiogenesis.

Non-peptidic Compounds are preferably used because peptidic vectors generally have poor biological stability become and unwanted Reactions of the body can provoke.

Prefers the vector is a compound that does not contain any unacceptable biological Reaction triggers, in particular a connection that actually does not promote angiogenesis.

Especially Preferably, the vector is an angiogenesis inhibitor, in particular preferably a non-peptidic angiogenesis inhibitor.

Examples of non-peptidic angiogenesis inhibitors are disclosed in U.S. Pat WO94 / 17084 (British Biotech), EP-A-618 208 (Daiichi), WO94 / 13277 (ICRT), WO95 / 06473 (Nippon Kayaku), WO94 / 21612 (Otsuka), WO97 / 37655 (Merck), WO97 / 30035 (Zeneca) EP-A-678 296 (Tsumura), WO94 / 18967 (Harvard), WO95 / 08327 (Dept. of Health and Human Services) (see also US-A-4590201 (Merck)) and EP-A-652000 (Eli Lilly).

Examples of peptidic angiogenesis inhibitors are disclosed in U.S. Pat WO94 / 02446 (British Biotech), WO94 / 02447 (British biotech), WO94 / 21625 (British Biotech), WO94 / 24140 (British Biotech), WO95 / 04033 (Celltech) EP-A-589 719 (Eli Lilly), US-A-5399667 (Frazier), EP-A-241830 (The General Hospital Corporation) and WO97 / 38009 (Merck).

Developmental angiogenesis inhibitors include those listed below in Table 3: Table 3 - Angiogenesis inhibitors connection target indications company Remarks Sodium tecogalan Kaposi's sarcoma, solid tumors Daiichi Sulfated polysaccharide peptidoglycan complex AGM-1470 Kaposi's sarcoma, malignant tumors Takeda / Abbott Fumagillin analog 00-01 Cancer metastasis Carbomed Polysaccharide exotoxin Mitoflaxon Solid tumors Lipha GM-1603 Glycomed Modified heparin rPF4 Kaposi's sarcoma, colon cancer, glioma, renal cell carcinoma, malignant melanoma Replistatin Repligen Recombinant Human Platelet Factor-4 MPF-4 Lilly Modified Human Platelet Factor-4 recombinant angiostatin EntreMed endostatin collagen fragment thalidomide Brain, thoracic and EntreMed Prostate Cancer DC101 ImClone monoclonal Systems antibody OLX 514 solid tumors, sepsis Aronex raloxifene Lilly Sodium suramin Metastatic hormone refractory prostate cancer Parke-Davis IL-12 kidney cancer Roche marimastat Pancreatic, lung and brain cancer British biotech CAI Wide range of cancers NCI Calcium channel blockers as well as the following peptic and non-peptidic drug compounds:

Other known compounds capable of targeting regions of angiogenesis are listed below in Table 4. Table 4 - Vector molecules with known affinity for receptors associated with angiogenesis vector molecules Angiopoietin angiostatin [plasminogen fragment] [inhibitor] Angiotensin II α2-antiplasmin combinatorial libraries, compounds of eg compounds that bind to the basal membrane after degradation β-cyclodextrin tetradecasulfate endoglin Endosialin Endostatin [collagen fragment] Epidermal growth factor (heparin-binding) Fibrin Fibrinopeptide B Fibroblast growth factor, FGF-3, basic Fibronectin Fumagillin and analogues Heparin Hepatocyte growth factor Hyaluronan Insulin-like growth factor Interferon-α, -β [inhibitors] Interleukins: IL- 8, IL-12 [inhibitor] laminin, laminin fragments leukemia inhibiting factor Linomide matrix metalloproteinase-2 metalloproteinases metalloproteinase inhibitors monoclonal antibodies for example: against angiogenic factors or their receptors or against components of the fibrinolytic system peptides: for example cyclic RGD ₀ FV Placental Growth Factor placental proliferin -related protein plasminogen plasminogen activator plasminogen activator inhibitor-1 antagonists of the platelet activating factor [inhibitors] platelet-derived growth factor (eg, type BB) platelet-derived growth factor receptors Platelet-derived Endothelial Cell Growth Factor Pleiotropin Proliferin, proliferin-related protein Selectins: E-selectin SPARC Snake Venome (RGD-containing) Substance P (a neuropeptide: neurokinin) Suramin tissue inhibitor of metalloproteinases (eg TIMP-2) Thalidomide Thrombin Thrombin receptor-activating Tetradecapeptide Transforming Growth Factor-α, -β Transforming Growth Factor Receptor Tumor Growth Factor-α Tumor Necrosis Factor Vitronectin Note: Many hormones, growth factors, and other compounds that bind to cell surface receptors can act as vectors by binding to their receptors, or, if already on the cell surface they are targets for vectors that bind to them, eg antibodies.

Similarly, the compounds that are in WO95 / 08327 can be used as vectors (see also Kohn et al., Proc. Nat. Acad Sci., USA 92: 1307-1311 (1995) and J. Clin. Oncol 15: 1985-1993 (1997)).

wobei
A für N oder CH steht; R¹ für H, -CONH, -CONHR⁵, COOH, -COOR⁵, SO₂NH₂ steht; R² für H, NH₂, NHCOPh, -NHCOR⁵, -NHCHO, -NHR⁵, -N(R⁵)₂ steht; und R⁵ ein Alkyl mit 1-6 Kohlenstoffatomen ist, z.B.Specific examples of vector compounds mentioned in some of the above-mentioned patent publications are as follows:
WO95 / 08327 (Dept. of Health and Human Services) describes angiogenesis inhibitor compounds of formulas I and II: Y- (CH ₂ ) _p -Ar ¹ -X-Ar ² (II) in which
Ar ¹ and Ar ^{2 are} aromatic groups and may be different or the same; and X is a linking group, for example, O, S, SO ₂ , CO, CHCN, alkyl, alkoxy or alkoxyalkyl, and

in which
A is N or CH; R ^{1 is} H, -CONH, -CONHR ⁵ , COOH, -COOR ⁵ , SO ₂ NH ₂ ; R ^{2 is} H, NH ₂ , NHCOPh, -NHCOR ⁵ , -NHCHO, -NHR ⁵ , -N (R ⁵ ) ₂ ; and R ^{5 is} an alkyl of 1-6 carbon atoms, eg

wobei
X für O, COO steht; und M ein Übergangsmetall ist; und

wobei
X₁ und X₂ substituierte oder unsubstituierte Phenyl- oder Naphthylgruppen sind; Y₁ und Y₂ Halogenatome, Aminogruppen oder mono- oder di-substituierte Aminogruppen sind; und Z für NHC₂H₄NH steht oder ein substituierter oder unsubstituierter aromatischer Diaminrest ist, z.B. WO95 / 06473 (Nippon Kayaku Kabushiki Kasai) discloses antitumor and angiogenesis inhibitor compounds of formulas 1 and 2:

in which
X is O, COO; and M is a transition metal; and

in which
X ₁ and X _{2 are} substituted or unsubstituted phenyl or naphthyl groups; Y ₁ and Y _{2 are} halogen atoms, amino groups or mono- or di-substituted amino groups; and Z is NHC ₂ H ₄ NH or is a substituted or unsubstituted aromatic diamine residue, eg

wobei R¹ =

R³ für H, Halogen oder CH₃, CF₃ oder OCH₃ steht; R² für H oder CH₃ steht, z.B. N⁴-Hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-4(chlorophenylpropyl)succinamid; N⁴-Hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methylphenylpropyl)succinamid; N⁴-Hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-methoxyphenylpropyl)succinamid; und N⁴-Hydroxy-N¹-(1-(S)-carbamoyl-2,2-dimethylpropyl)-2-(R)-(4-trifluormethylphenylpropyl)succinamid. WO95 / 04033 (Celltech Limited) discloses the following angiogenesis inhibitors:

where R ¹ =

R ^{3 is} H, halogen or CH ₃ , CF ₃ or OCH ₃ ; R ^{2 is} H or CH ₃ , for example N ⁴ -hydroxy-N ¹ - (1- (S) -carbamoyl-2,2-dimethylpropyl) -2- (R) -4 (chlorophenylpropyl) succinamide; N ⁴ -hydroxy-N ¹ - (1- (S) -carbamoyl-2,2-dimethylpropyl) -2- (R) - (4-methylphenylpropyl) succinamide; N ⁴ -hydroxy-N ¹ - (1- (S) -carbamoyl-2,2-dimethylpropyl) -2- (R) - (4-methoxyphenylpropyl) succinamide; and N ⁴ -hydroxy-N ¹ - (1- (S) -carbamoyl-2,2-dimethylpropyl) -2- (R) - (4-trifluoromethylphenylpropyl) succinamide.

EP 241830 (The General Hospital Corporation) discloses the purification of Hepatoma-derived Growth Factor (HDGF), which is an endothelial mitogen and a powerful angiogenic factor. The use of HDGF in controlling angiogenesis and detecting cancerous liver tumors by using an immunodiagnostic assay is also described. The HDGF peptide fragment has an N-terminal amino acid sequence wherein the first 16 amino acids are:
leu-pro-ala-leu-pro-glu-asp-gly-gly-xx-gly-ala-phe-pro-pro-gly
(xx = unidentified amino acid unit).

An HDGF peptide fragment is also disclosed having an N-terminal amino acid extension sequence comprising:
(Ala / ser) - (leu / arg) -Pro- (ala / gly) - (leu / pro) -ala-gly-thr-met-ala- (ala) -Gly-Ser (isoleu) -Thr- thr-leu

wobei
R¹ und R³ für H, Me, -C(O)(C₁-C₆ Alkyl), -C(O)Ar stehen; Ar optional substituiertes Phenyl ist; und R² Pyrrolidino oder Piperidino ist, z.B. EP 652000 (Eli Lilly and Company) discloses angiogenesis inhibitor and inhibitor compounds of angiogenic diseases having the formula:

in which
R ¹ and R ^{3 are} H, Me, -C (O) (C ₁ -C ₆ alkyl), -C (O) Ar; Ar is optionally substituted phenyl; and R ^{2 is} pyrrolidino or piperidino is, for example

EP 652000 [589719?] (Eli Lilly and Company) discloses a modified Platelet Factor-4 having the amino acid sequence:

MPF-4

• NH ₂ -Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln -Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Lys-Ile-Ile-Lys -Lys-Leu-Leu-Glu-Ser-COOH

CPF-4

• NH ₂ -Ser-Gln-Val-Arg-Pro-Arg-His-Ile-Thr-Ser-Leu-Glu-Val-Ile-Lys-Ala-Gly-Pro-His-Cys-Pro-Thr-Ala-Gln -Leu-Ile-Ala-Thr-Leu-Lys-Asn-Gly-Arg-Lys-Ile-Cys-Leu-Asp-Leu-Gln-Ala-Pro-Leu-Tyr-Lys-Ile-Ile-Lys-Lys Leu-Leu-Glu-Ser-COOH disulfide bound to a second protein having the amino acid sequence NH ₂ -Glu-Ala-Glu-Glu-Asp-Gly-Asp-Leu-Gln-Cys-Leu-Cys-Val Lys-Thr-Thr-COOH. There are disulfide bonds between Cys-20 of MPF-4 and Cys-10 of said second protein and Cys-36 of MPF-4 and Cys-12 of said second protein.

wobei
R¹ bis R⁴ jeweils unabhängig für eines oder mehrere von -X, N₃, -NO₂, Halo, Trifluormethyl, R⁵, OR⁵, -CH₂OR⁶, -OCOR⁵, -CH₂COOR⁵, -NHCOR⁵, -CH₂NHCOR⁵, -NR⁵R⁶, -CH₂NR⁵R⁶, -CH₂NO₂, CONR⁵R⁶, CH₂CONR⁵R⁶, -COOR⁵, -CH₃COOR⁵, -CHO und CH₂CHO steht und -X unabhängig für -SO₃R⁵, -CH₂PO₃R⁵R⁶, -CH₂O₃R⁵, -OSO₃R⁵, -CH₂OSO₃R⁵, -NHSO₃R⁵, -CH₂NHSO₃R⁵, OPO₃R⁵R⁶, -CH₂OPO₃R⁵R⁶ und -PO₃R⁵R⁶ steht, wobei R⁵ und R⁶ unabhängig aus -H und Niederalkyl ausgewählt sind und wobei A eine chemische Gruppe ist, die mindestens 5 bis nicht mehr als 30 Bindungen umfasst, die die Naphthylgruppen direkt verbinden, vorausgesetzt, dass (i) die Verbindung nicht Suramin ist und (ii) wenn A nicht

ist, wobei
m und n unabhängig 0, 1 oder 2 sind, dann mindestens eine von R¹ bis R⁴ für -OH oder eine saure Gruppe steht; und der pharmazeutisch annehmbaren Salze, Ester, Salze derartiger Ester oder Amide derartiger Verbindungen. WO94 / 13277 (Imperical Cancer Research Technology Limited) discloses the use of compounds of formula I:

in which
R ¹ to R ^{4 are} each independently one or more of -X, N ₃ , -NO ₂ , halo, trifluoromethyl, R ⁵ , OR ⁵ , -CH ₂ OR ⁶ , -OCOR ⁵ , -CH ₂ COOR ⁵ , -NHCOR ⁵ , -CH ₂ NHCOR ⁵ , -NR ⁵ R ⁶ , -CH ₂ NR ⁵ R ⁶ , -CH ₂ NO ₂ , CONR ⁵ R ⁶ , CH ₂ CONR ⁵ R ⁶ , -COOR ⁵ , -CH ₃ COOR ⁵ , -CHO and CH ₂ CHO and -X independently represent -SO ₃ R ⁵ , -CH ₂ PO ₃ R ⁵ R ⁶ , -CH ₂ O ₃ R ⁵ , -OSO ₃ R ⁵ , -CH ₂ OSO ₃ R ⁵ , -NHSO ₃ R ⁵ , -CH ₂ NHSO ₃ R ⁵ , OPO ₃ R ⁵ R ⁶ , -CH ₂ OPO ₃ R ⁵ R ⁶ and -PO ₃ R ⁵ R ⁶ , where R ⁵ and R ^{6 are} independently -H and lower alkyl and wherein A is a chemical group comprising at least 5 to not more than 30 bonds connecting the naphthyl groups directly, provided that (i) the compound is not suramin and (ii) when A is not

is, where
m and n are independently 0, 1 or 2, then at least one of R ¹ to R ^{4 is} -OH or an acidic group; and the pharmaceutically acceptable salts, esters, salts of such esters or amides of such compounds.

Compounds are also described in which the connection of A to the naphthyl ring takes place via an amide or sulfonamide group. In addition, A may in some cases be a group of formula II. A may also be selected from straight chain or branched alkyl groups, aryl groups, alkylaryl groups, aliphatic dicarboxylic acids, polynes and derivatives thereof, and polyols and derivatives thereof. Some other compounds have the formulas:

z.B. EP 678296 (Tsumura & Co.) discloses angiogenesis inhibitors of the general formulas:

eg

WO94 / 18967 (President and Fellows of Harvard College) discloses a class of imidazoles that inhibit angiogenesis:

z.B. EP-A-618 208 (Daiichi Pharmaceutical) discloses compounds of the formula:

eg

z.B. WO94 / 02446 (British Biotechnology Limited) discloses compounds of the formula:

eg

z.B. WO94 / 02447 (British Biotechnology Limited) discloses compounds of the formula:

eg

z.B. WO 94/21625 (British Biotechnology Limited) discloses compounds of the formula:

eg

wobei
X für -CONHOH oder COOH steht, in erster Linie gekennzeichnet durch das Vorhandensein einer Gruppe der Formel (II) im Substituenten R³ und/oder R⁴

z.B. WO94 / 24149 (British Biotechnology Limited) discloses compounds of formula (I):

in which
X is -CONHOH or COOH, characterized primarily by the presence of a group of the formula (II) in the substituent R ³ and / or R ⁴

eg

Particularly preferred vectors include amino acid derivatives, as in WO94 / 02446 described, hydroxamic acid derivatives, as in WO94 / 02447 described, Thiazolpyrimidines, as in EP-A-618 208 described triazoles, as in WO95 / 08327 described, quinazolines, as in WO97 / 30035 described, isoindolone, as in WO97 / 37655 described, integrin inhibitors, VEGF antagonists, bFGF antagonists, thrombospondin and thrombospondin fragments, CD36 and growth factors (eg VEGF, bFGF, etc.).

CAM-D and other eligible identification and evaluation techniques as mentioned above can also used to further eligible peptidic and non-peptidic Find or rate vectors.

Therefore it is also possible molecules specifically to angiogenesis-associated receptors bind, by directly screening molecular libraries. screening Peptidic libraries can also be used to generalize to identify effective peptidic structures, of which non-peptidic Analogues by conventional or combinatorial chemistry can be generated. Bonding units, the Identified in this way can be coupled to a linker molecule become what a common tool for tying any vector molecule (or molecules) to the reporter.

Vector molecules can out combinatorial libraries are generated, without necessarily the to know the exact molecular target by functional selection (in vitro, ex vivo or in vivo) of molecules attached to the region (s) Structure to be imaged bind.

As mentioned above, the agents of formula I include vector, linker and reporter units. A linker unit may serve to link a vector to a reporter; alternatively, it may connect more than one vector and / or more than one reporter. Similarly, a reporter or vector can be linked to more than one linker. The use of a plurality of reporters in this manner (eg multiple linker reporter moieties linked to one vector or multiple reporters bound to a linker which itself is linked to a vector) may allow the detectability of the contrast agent to be increased (eg Increasing radiopacity, echogenicity or relaxivity) or may allow it to be detected in more than one imaging modality. Using a plurality of vectors in this manner may increase the targeting efficiency of the contrast agent or may enable the contrast agent to target more than one site, eg, different receptors to target an agent that has receptor heterogeneity. Thus, for example, the agent of formula I may comprise vector units having affinity sites other than angiogenesis-associated receptors, eg, with affinities for tent surfaces on body duct wall surfaces. Accordingly, the agent may comprise vectors such as antibody fragments and oligopeptides, eg RGD or analogous cell surface binding peptide motifs (eg as described in U.S. Pat EP-A-422937 and EP-A-422 938 (Merck)) or other vectors, as in GB 9700699.3 described. Such additional vectors may also be selected from any of the molecules that naturally concentrate in a selected target organ, tissue, cell or group of cells or elsewhere in a mammalian body in vivo. These may include amino acids, oligopeptides (eg hexapeptides), Molecular Recognition Units (MRUs), single chain antibodies (SCAs), proteins, non-peptidic organic molecules, Fab fragments and antibodies. Examples of site-directed molecules include polysaccharides (eg, CCK and hexapeptides), proteins (such as lectins, asialofetuin, polyclonal IgG, blood clotting proteins (eg, hirudin), lipoproteins, and glycoproteins), hormones, growth factors, coagulation factors (such as PF4), polymerized fibrin fragments (eg, E ₁ ), Serum amyloid precursor (SAP) proteins, low density lipoprotein (LDL) precursors, serum albumin, intact red blood cell surface proteins, receptor binding molecules such as estrogens, liver specific proteins / polymers such as galactosyl neoglycoalbumin (NGA) (see Vera et al Radiology 151: 191 (1984)) N- (2-hydroxypropyl) methacrylamide (HMPA) copolymers with varying numbers of bound galactosamines (see Duncan et al., Biochem. Biophys. Acta 880: 62 (1986)) and allyl and 6 Aminohexyl glycosides (see Wong et al., Carbo. Res. 170: 27 (1987)) and fibrinogen. The site-directed protein may also be an antibody. The choice of antibody, particularly the antigenic specificity of the antibody, will depend on the particular intended target site for the agent. Monoclonal antibodies are preferred over polyclonal antibodies. The production of antibodies that react with a desired antigen is well known. Antibody preparations are commercially available from a variety of sources. Fibrin fragment E ₁ can be prepared as described by Olexa et al. in J. Biol. Chem. 254: 4925 (1979). The production of LDL precursors and SAP proteins is described by de Beer et al. in J. Immunol. Methods 50:17 (1982).

It it is particularly preferred that such additional vectors bind in such a way should slow down the movement of the agent in the blood stream, but do not prevent it, and anchor it in place when it is at one Receptor site associated with angiogenesis is bound.

functional Groups (e.g., amino groups, hydroxyl groups, carboxy groups, thiol groups, etc.) on the vector compound for binding the vector to the linker unit or directly to the reporter unit can be used, e.g. using conventional chemical Coupling techniques.

Where the vector is a peptidic compound, the reporter is one Multireporter, e.g. a metalated polychelant (preferred a dendrimeric polychelant), a magnetic (preferred superparamagnetic) particle, a vesicle, the Particles or a solution a contrasting molecule contains a polyionic species (e.g., a polymer containing a variety of ionic Groups, preferably anionic groups, e.g. a carboxylate, phosphate or sulfonate polymer).

Where the vector is nonpeptidic, the reporter may be a multireporter or alternatively have one or a small number (eg, up to 10) detectable markers, eg, chelated paramagnetic metal ions, covalently bound or chelated radioisotopes, and chromophores (or fluorophores, etc.). , Where the reporter is or has a covalently bound radionuclide, this is an iodine radionuclide preferred over a tritium or ¹³ C atom.

left

A Wide variety of linkers can be used, including biodegradable ones Linker and biopolymers.

The Linker component of the contrast agent is most easily a bond between the vector and reporters units. More general is the Left, however, a mono- or to provide a multimolecular skeleton that is covalent or non-covalent associates one or more vectors with one or more reporters, e.g. a linear, cyclic, branched or crosslinked molecular Skeleton, or a molecular aggregate, with built-in or lateral Groups which are covalently or non-covalently, e.g. coordinative, to the vector and reporter units bind or such units encapsulate, capture or anchor.

Therefore, joining a reporter moiety to a desired vector can be accomplished by covalent or non-covalent means, usually involving interaction with one or more functional groups arranged on the reporter and / or vector is involved. Examples of chemically reactive functional groups that can be used for this purpose include amino, hydroxyl, sulfhydryl, carboxyl and carbonyl groups, as well as carbohydrate groups, vicinal diols, thioethers, 2-amino alcohols, 2-aminothiols, guanidinyl, Imidazolyl and phenolic groups.

Covalent coupling of reporter and vector can therefore be effected using linking agents containing reactive moieties capable of reaction with such functional groups. Examples of reactive moieties capable of reaction with sulfhydryl groups include alpha-haloacetyl compounds of the type X-CH ₂ CO- (where X = Br, Cl or I), which exhibit particular reactivity for sulfhydryl groups, but which may also be used to modify imidazolyl, thioether, phenol and amino groups as described by Gurd, FRN in Methods Enzymol. (1967) 11, 532. N-maleimide derivatives may also be considered selective to sulfhydryl groups, but may also be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane, eg as described by Traut, R. et al. in Biochemistry (1973) 12, 3266, which introduce a thiol group by conversion of an amino group can be considered as sulfhydryl reagents, if joining occurs through the formation of disulfide bridges. Thus, reagents that introduce reactive disulfide bonds into either the reporter or the vector may be useful, as the joining can be induced by disulfide interchange between the vector and the reporter; Examples of such reagents include Ellman's reagent (DTNB), 4,4'-dithiodipyridine, methyl 3-nitro-2-pyridyl disulfide, and methyl 2-pyridyl disulfide (described by Kimura, T., et al., Analyt. Biochem ) 122, 271).

• i) α-Haloacetylverbindungen, die eine Spezifität gegenüber Aminogruppen in Abwesenheit reaktiver Thiolgruppen zeigen und vom Typ X-CH₂CO- sind (wobei X=Cl, Br oder I), z.B. wie beschrieben von Wong, Y-H.H. in Biochemistry (1979) 24, 5337;
• ii) N-Maleimidderivate, die mit Aminogruppen entweder über eine Reaktion des Michael-Typs oder über Acylierung durch Addition an die Ringcarbonylgruppe reagieren können, wie von Smyth, D.G. et al. in J. Am. Chem. Soc. (1960) 82, 4600 und Biocher. J. (1964) 91, 589, beschrieben;
• iii) Arylhalogenide, wie reaktive nitrohaloaromatische Verbindungen;
• iv) Alkylhalogenide, wie von McKenzie, J.A. et al. in J. Protein Chem. (1988) 7, 581 beschrieben;
• v) Aldehyde und Ketone, die der Schiff-Basenbildung mit Aminogruppen fähig sind, wobei die gebildeten Addukte üblicherweise über eine Reduktion stabilisiert werden, um ein stabiles Amin zu ergeben;
• vi) Epoxidderivate, wie Epichlorohydrin und Bisoxirane, die mit Amino-, Sulfhydryl- oder Phenolhydroxylgruppen reagieren können;
• vii) Chlor enthaltende Derivate von s-Triazinen, die gegenüber Nukleophilen, wie Amino-, Sulfhydryl- und Hydroxygruppen, sehr reaktiv sind; viii) Aziridine, die auf oben im Detail angegebenen s-Triazinverbindungen basieren, z.B. wie beschrieben von Ross, W.C.J. in Adv. Cancer Res. (1954) 2, 1, die mit Nukleophilen, wie Aminogruppen, durch Ringöffnung reagieren;
• ix) Quadratsäurediethylester, wie von Tietze, L.F. in Chem. Ber. (1991) 124, 1215 beschrieben; und
• x) α-Haloalkylether, die reaktivere Alkylierungsmittel als normale Alkylhalogenide sind wegen der Aktivierung, die durch das Ethersauerstoffatom verursacht sind, z.B. wie beschrieben von Benneche, T. et al. in Eur. J. Med. Chem. (1993) 28, 463.

Examples of reactive moieties capable of reacting with amino groups include alkylating and acylating agents. Representative alkylating agents include:

• i) α-haloacetyl compounds which exhibit specificity to amino groups in the absence of reactive thiol groups and are of the X-CH ₂ CO- type (where X = Cl, Br or I), for example as described by Wong, YH.H. in Biochemistry (1979) 24, 5337;
• ii) N-maleimide derivatives which can react with amino groups either via a Michael-type reaction or via acylation by addition to the ring carbonyl group as described by Smyth, DG et al. in J. Am. Chem. Soc. (1960) 82, 4600 and Biocher. J. (1964) 91, 589;
• iii) aryl halides, such as reactive nitrohaloaromatic compounds;
• iv) alkyl halides as described by McKenzie, JA et al. in J. Protein Chem. (1988) 7, 581;
• v) aldehydes and ketones capable of Schiff base formation with amino groups, the adducts formed usually being stabilized via reduction to give a stable amine;
• vi) epoxide derivatives such as epichlorohydrin and bisoxiranes capable of reacting with amino, sulfhydryl or phenolic hydroxyl groups;
• vii) chlorine-containing derivatives of s-triazines which are highly reactive towards nucleophiles such as amino, sulfhydryl and hydroxy groups; viii) aziridines based on s-triazine compounds detailed above, eg as described by Ross, WCJ in Adv. Cancer Res. (1954) 2,1, which react with nucleophiles such as amino groups by ring opening;
• ix) diethyl squarate, as described by Tietze, LF in Chem. Ber. (1991) 124, 1215; and
• x) α-haloalkyl ethers which are more reactive alkylating agents than normal alkyl halides because of activation caused by the ether oxygen atom, eg as described by Benneche, T. et al. in Eur. J. Med. Chem. (1993) 28, 463.

• i) Isocyanate und Isothiocyanate, insbesondere aromatische Derivate, die stabile Harnstoff- bzw. Thioharnstoffderivate bilden und für die Proteinvernetzung verwendet wurden, wie von Schick, A.F. et al. in J. Biol. Chem. (1961) 236, 2477 beschrieben;
• ii) Sulfonylchloride, die von Herzig, D.J. et al. in Biopolymers (1964) 2, 349 beschrieben wurden, und die für die Einführung einer fluoreszierenden Reportergruppe in den Linker nützlich sein können,
• iii) Säurehalogenide;
• iv) aktive Ester, wie Nitrophenylester oder N-Hydroxysuccinimidylester;
• v) Säureanhydride, wie gemischte, symmetrische oder N-Carboxyanhydride;
• vi) andere nützliche Reagenzien für die Amidbindungsbildung, wie von Bodansky, M. et al. in "Principles of Peptide Synthesis" (1984) Springer-Verlag beschrieben;
• vii) Acylazide, z.B. in denen die Azidgruppe aus einem vorgebildeten Hydrazidderivat unter Verwenden von Natriumnitrit erzeugt wird, z.B. wie beschrieben von Wetz, K. et al. in Anal. Biochem. (1974) 58, 347;
• viii) Azlactone, gebunden an Polymere, wie Bisacrylamid, z.B. wie von Rasmussen, J.K. in Reactive Polymers (1991) 16, 199 beschrieben; und
• ix) Imidoester, die stabile Amidine bei der Reaktion mit Aminogruppen bilden, z.B. wie beschrieben von Hunter, M.J. und Ludwig, M.L. in J. Am. Chem. Soc. (1962) 84, 3491.

Representative amino-reactive acylating agents include:

• i) isocyanates and isothiocyanates, in particular aromatic derivatives which form stable urea or thiourea derivatives and were used for protein crosslinking, as described by Schick, AF et al. in J. Biol. Chem. (1961) 236, 2477;
• ii) sulfonyl chlorides described by Herzig, DJ et al. in Biopolymers (1964) 2, 349, and which may be useful for the introduction of a fluorescent reporter group into the linker,
• iii) acid halides;
• iv) active esters, such as nitrophenyl esters or N-hydroxysuccinimidyl esters;
• v) acid anhydrides, such as mixed, symmetrical or N-carboxyanhydrides;
• vi) other useful reagents for amide bond formation as described by Bodansky, M. et al. in "Principles of Peptide Synthesis" (1984) Springer-Verlag;
• vii) acylazides, eg in which the azide group is generated from a preformed hydrazide derivative using sodium nitrite, eg as described by Wetz, K. et al. in anal. Biochem. (1974) 58, 347;
• viii) azlactones bonded to polymers such as bisacrylamide, for example as described by Rasmussen, JK in Reactive Polymers (1991) 16, 199; and
• ix) Imidoesters which form stable amidines in the reaction with amino groups, for example as described by Hunter, MJ and Ludwig, ML in J. Am. Chem. Soc. (1962) 84, 3491.

carbonyl groups, like aldehyde functions, can with weak protein bases at such a pH for reaction be brought that the nucleophilic protein side chain functions be protonated. Weak bases include 1,2-aminothiols, such as those in N-terminal ones Cysteine residues are found that selectively stably 5-membered Forming thiazolidine rings with aldehyde groups, e.g. as described by Ratner, S. et al. in J. Am. Chem. Soc. (1937) 59, 200. Others weak bases such as phenylhydrazones may be used, e.g. as described by Heitzman, J. et al. in proc. Natl. Acad. Sci. USA (1974) 71, 3537.

aldehydes and ketones can can also be reacted with amines to ship bases form that advantageously over reductive amination can be stabilized. Alkoxylaminoeinheiten react easily with ketones and aldehydes to form stable alkoxamines to produce, e.g. as reported by Webb, R. et al. in Bioconjugate Chem. (1990) 1, 96.

Examples reactive moieties capable of reaction with carboxyl groups, include diazo compounds such as diazoacetate esters and diazoacetamides, those with high specificity react to produce ester groups, e.g. as described by Herriot R.M. in Adv. Protein Chem. (1947) 3, 169. Carboxylic acid modifying Reagents, such as carbodiimides, which undergo O-acyl urea formation, followed by amide bond formation can also be usefully used; the Joining can be simplified by adding an amine or can lead to a direct vector receptor coupling. Useful water-soluble carbodiimides include 1-cyclohexyl-3- (2-morpholinyl-4-ethyl) carbodiimide (CMC) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), e.g. as by Zot, H.G. and Puett, D. in J. Biol. Chem. (1989) 264, 15552 described. Other useful carboxylic acid Modifying reagents include isoxazolium derivatives, such as Woodward's reagent K; Chloroformates, such as p-nitrophenyl chloroformate, carbonyldiimidazoles, such as 1,1'-carbonyldiimidazole; and N-carbalkoxydihydroquinolines such as N- (ethoxycarbonyl) -2-ethoxy-1,2-dihydroquinoline.

Other possibly useful reactive moieties include vicinal diones such as p-phenylene diglyoxal, which can be used to react with guanidinyl groups, e.g. as described by Wagner et al. in Nucleic Acid Res. (1978) 5, 4065; and diazonium salts, which can be subjected to electrophilic substitution reactions, e.g. as reported by Ishizaka, K. and Ishizaka, T. in J. Immunol. (1960) 85, 163 described. Bis-diazonium compounds can easily be treated of aryl diamines with sodium nitrite in acidic solutions. It will be recognized that functional groups in the reporter and / or vector, if desired is converted to other functional groups before the reaction can be e.g. for an additional Reactivity or selectivity to rent. Examples of methods that are useful for this purpose include the conversion of amines to carboxylic acids using reagents, such as dicarboxylic anhydrides; the conversion of amines to thiols using reagents, such as N-acetyl homocysteine thiolactone, S-acetylmercaptosuccinic anhydride, 2-iminothiolane or thiol-containing succinimidyl derivatives; the Conversion of thiols to carboxylic acids using reagents, such as α-haloacetates; the conversion of thiols to amines using reagents such as Ethyleneimine or 2-bromoethylamine; the conversion of carboxylic acids too Amines are followed using reagents such as carbodiimides of diamines; and converting alcohols to thiols using of reagents such as tosyl chloride, followed by transesterification with thioacetate and hydrolysis to the thiol with sodium acetate.

The Vector reporter coupling can also be effected using enzymes such as zero-length crosslinkers; therefore, e.g. Transglutaminase, peroxidase and xanthine oxidase are used to make crosslinked products to create. Reverse proteolysis can also be used to crosslink via amide bond formation.

Non-covalent Vector reporter coupling can be done, for example, by electrostatic Charge interactions are effected via chelation in the mold stable metal complexes or over High affinity binding interaction.

One Vector attached to a peptide, lipo-oligosaccharide or lipopeptide linker which contains an element capable of incorporating a membrane can also be useful be. An example is given by Leenhouts, J.M. et al. in Feb's Letters (1995) 370 (3), 189-192.

The coupling can also be effected using avidin or streptavidin, which have four high affinity binding sites for biotin. Avidin can therefore be used to conjugate a vector to a reporter if both the vector and the reporter are biotinylated. Examples will be by Bayer, EA and Wilchek, M. in Methods Biochem. Anal. (1980) 26, 1. This method can also be extended to include linking the reporter to a reporter, a process that promotes association of the agent and consequent increased potency. Alternatively, avidin or streptavidin can be bound directly to the surface of reporter particles.

Noncovalent coupling may also exploit the bifunctional nature of bispecific immunoglobulins. These molecules can specifically bind two antigens and bind them together. For example, either bispecific IgG or chemically generated bispecific F (ab) ' ₂ fragments may be used as the linking agent. Heterobifunctional bispecific antibodies have also been reported for joining two different antigens, eg as described by Bode, C. et al. in J. Bio. Chem. (1989) 264, 944 and by Staerz, UD et al. in proc. Natl. Acad. Sci. USA (1986) 83, 1453. Similarly, any reporter and / or vector possessing two or more antigenic determinants (eg as described by Chen, Aa et al., Am. J. Pathol. (1988) 130, 216 be crosslinked by antibody molecules and lead to the formation of crosslinked accumulations of agents of the formula I of a possibly increased efficacy.

So-called Zero-length bonding agent, which is a direct covalent bond two reactive chemical groups without the introduction of additional induce binding (e.g., as in amide bond formation, which induced using carbodiimides or enzymatically will), if desired, in accordance used with the invention, as well as many agents, such as biotin / avidin systems, which induce a non-covalent reporter vector compound and agents, induce electrostatic interactions.

in the However, in the most general case, the connecting means becomes two or more have more reactive units, e.g. as described above through a spacer element. The presence of such a spacer allows bifunctional linkers, with specific functional Groups within a molecule or to react between two different molecules, resulting in one Binding between these two components and inserting extrinsic linker-derived material into the reporter-vector conjugate. The reactive moieties in a linking agent may be the same (homobifunctional Means) or various (heterobifunctional agents or where several dissimilar reactive units are present, heteromultifunctional agents) be, leading to a variety of possible Leads reagents, the one covalent bond between any chemical species either intramolecular or intermolecular can cause.

The Nature of extrinsic material introduced by the bonding agent, may be of critical concern to the targeting ability and general stability be the final product. Therefore, it may be desirable to have labile compounds introduce, e.g. contain the spacer arms, which biodegradable or chemical sensitive, or the enzymatic cleavage sites incorporated to have. Alternatively, the spacer may comprise polymeric components, e.g. as a surface-active To act agents and to improve the stability of the agent. The spacer may also contain reactive moieties, e.g. as above described the surface crosslinking to improve.

spacer elements can typically consist of aliphatic chains containing the reactive Units of the linker over Separate distances between 5 and 30 Å. You can also contain macromolecular structures, such as poly (ethylene) glycols. Such polymeric structures, hereinafter referred to as PEGs, are simple, neutral polyethers used in biotechnical and biomedical Applications great Attention has been paid (see, e.g., Milton Harris, J. (ed.)) "Poly (ethylene glycol)" chemistry, biotechnical and biomedical applications ", Plenum Press, New York, 1992). PEGs are in most solvents including water soluble and are in watery Environments strongly hydrated, with two or three water molecules attached to each Be bound ethylene glycol segment; this has the effect of preventing it adsorption of either other polymers or proteins to the PEG-modified surfaces. PEGs are for it known to be non-toxic and active proteins or cells do not harm while covalent bound PEGs for it are known to be non-immunogenic and non-antigenic. In addition, PEGs slightly modified and to other molecules with little effect be tied to their chemistry. Your beneficial solubility and biological properties are among the many possible uses of PEGs and the associated ones Copolymers, including Block copolymers, such as PEG polyurethanes and PEG polypropylenes apparently.

suitable Molecular weights for PEG spacers in agreement can be used with the invention, for example, between 120 Daltons and 20 kDaltons lie.

Of the Main mechanism for the uptake of particles by the cells of the reticuloendothelial system (RES) is the opsonization by plasma proteins in the blood; these mark foreign particles, which are then taken up by the RES. The biological properties of PEG spacer elements in agreement can be used with the invention can serve the circulation time of the agent on a similar one Way to increase like those for PEGylated liposomes was observed (see, e.g., Klibanov, A.L. et al. in FEBS Letters (1990) 268, 235-237 and Blume, G. and Cevc, G. in Biochim. Biophys. Acta (1990) 1029, 91-97). An increased coupling efficiency Areas of interest can also be achieved using antibodies which are attached to the termini of PEG spacers (see e.g. Maruyama, K. et al. in Biochim. Biophys. Acta (1995) 1234, 74-80 and Hansen, C.B. et al. in Biochim. Biophys. Acta (1995) 1239, 133-144).

Other representative Spacer elements include structure-type polysaccharides such as polygalacturonic acid, glycosaminoglycans, Heparinoids, cellulose and marine polysaccharides, such as alginates, Chitosans and carrageenans; Memory type polysaccharides, such as Strength, Glycogen, dextran and aminodextrans, polyamino acids and methyl and ethyl esters of which, as in homo- and copolymers of lysine, glutamic acid and aspartic acid; and polypeptides, oligosaccharides and oligonucleotides, the enzyme cleavage sites can contain or not.

In general, spacer elements may contain cleavable groups such as vicinal glycol, azo, sulfone, ester, thioester or disulfide groups. Spacer, the biodegradable methylene diester or diamide groups of the formula - (Z) _m .YXC (R ¹ R ² ) .XY (Z) _n [wherein X and Z are -O-, -S- and -NR- (wherein R is hydrogen or an organic group) are selected; each Y is a carbonyl, thiocarbonyl, sulfonyl, phosphoryl or similar acid-forming group; m and n are each zero or 1; and R ¹ and R ^{2 are} each hydrogen, an organic group or a group -XY (Z) _m - or together form a divalent organic group] may also be useful; like in WO-A-9217436 however, such groups are readily biodegraded in the presence of esterases, eg, in vivo, but are stable in the absence of such enzymes. They may therefore be advantageously combined with therapeutic agents to facilitate their slow release.

Poly [N- (2-hydroxyethyl) methacrylamides] may be useful Spacer materials thanks to their low degree of interaction with Cells and tissues (see, e.g., Volfová, I., Rihová, B. and V. R., and Vetvicka, P. in J. Bioact. Comp. Polymers (1992) 7, 175-190). A work about a similar Polymer, mainly from the closely related 2-hydroxypropyl derivative, that it is only in a fairly wide range due to the mononuclear phagocytic system small extent Endocytosis was recorded (see Goddard, P., Willamson, I., Bron, J., Hutchkinson, L.E., Nicholls, J. and Petrak, K. in J. Bioct. Compat. Polym. (1991) 6, 4-24).

• i) Copolymere von Methylmethacrylat mit Methacrylsäure; diese können erodierbar sein (siehe Lee, P.I. in Pharm. Res. (1993) 10, 980) und die Carboxylatsubstituenten können einen höheren Grad an Ausdehnung als mit neutralen Polymeren verursachen;
• ii) Blockcopolymere von Polymethacrylaten mit bioabbaubaren Polyestern (siehe z.B. San Roman, J. und Guillen-Garcia, P. in Biomaterials (1991) 12, 236-241);
• iii) Cyanoacrylate, d.h. Polymere von Estern von 2-Cyanoacrylsäure – diese sind bioabbaubar und wurden in der Form von Nanoteilchen für selektive Arzneimittelverabreichung verwendet (siehe Forestier, F., Gerrier, P., Chaumard, C., Quero, A.M., Couvreur, P. und Labarre, C. in J. Antimicrob. Chemoter. (1992) 30, 173-179);
• iv) Polyvinylalkohole, die wasserlöslich sind und allgemein als biokompatibel angesehen werden (siehe z.B. Langer, R. in J. Control. Release (1991) 16, 53-60);
• v) Copolymere von Vinylmethylether mit Maleinsäureanhydrid, von denen festgestellt wurde, dass sie bioerodierbar sind (siehe Finne, U., Hannus, M. und Urtti, A., in Int. J. Pharm. (1992) 78, 237-241);
• vi) Polyvinylpyrrolidone, z.B. mit einem Molekulargewicht, das geringer als ungefähr 25.000 ist, die schnell durch die Nieren gefiltert werden (siehe Hespe, W., Meier, A. M. und Blankwater, Y.M. in Arzheim.-Forsch./Drug Res. (1977) 27, 1158-1162);
• vii) Polymere und Copolymere von kurzkettigen aliphatischen Hydroxysäuren, wie Glykol-, Milch-, Butter-, Valerian- und Capronsäuren (siehe z.B. Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9), einschließlich Copolymere, die aromatische Hydroxysäuren einbauen, um ihre Abbaugeschwindigkeit zu erhöhen (siehe Imasaki, K., Yoshida, M., Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H. und Nagai. T. in Int. J Pharm. (1992) 81, 31-38);
• viii) Polyester, die aus alternierenden Einheiten von Ethylenglykol und Terephthalsäure bestehen, z.B. Dacron^R, die nicht abbaubar sind, aber hoch biokompatibel;
• ix) Blockcopolymere, die bioabbaubare Segmente von aliphatischen Hydroxysäurepolymeren umfassen (siehe Younes, H., Nataf, P.R., Cohn, D., Appelbaum, Y.J., Pizov, G. und Uretzky, G. in Biomater. Artif Cells Artif. Organs (1988) 16, 705-719), beispielsweise in Zusammenhang mit Polyurethanen (siehe Kobayashi, H., Hyon, S.H. und Ikada, Y. in "Watercurable and biodegradable prepolymers" – J. Biomed. Mater. Res. (1991) 25, 1481-1494);
• x) Polyurethane, die als gut toleriert in Implantaten bekannt sind und die kombiniert werden können mit flexiblen "weichen" Segmenten, z.B. die Poly(tetramethylenglykol), Poly(propylenglykol) oder Poly(ethylenglykol) umfassen, und aromatischen "harten" Segmenten, z.B. die 4,4'-Methylenbis(phenylenisocyanat) umfassen (siehe z.B. Ratner, B.D., Johnston, A.B. und Lenk, T.J. in J. Biomed. Mater. Res: Applied Biomaterials (1987) 21, 59-90; Sa Da Costa, V. et al. in J. Coll. Interface Sci. (1981) 80, 445-452 und Affrossman, S. et al. in Clinical Materials (1991) 8, 25-31);
• xi) Poly(1,4-dioxan-2-one), die als bioabbaubare Ester angesichts ihrer hydrolysierbaren Esterverbindungen betrachtet werden können (siehe z.B. Song, C. X., Cui, X.M., und Schindler, A. in Med. Biol. Eng. Comput. (1993) 31, S147-150), und Glycolideinheiten umfassen können, um ihre Absorptionsfähigheit zu verbessern (siehe Bezwada, R.S., Shalaby, S.W. und Newman, H.D.J. in Agricultural and synthetic polymers: Biodegradability and utilization (1990) (Hrsg. Glass, J.E. und Swift, G.), 167-174 – ACS Symposium Series, #433, Washington D.C., U.S.A. – American Chemical Society);
• xii) Polyanhydride, wie Copolymere von Sebacinsäure (Octandisäure) mit bis(4-Carboxyphenoxy)propan, für die in Studien mit Kaninchen (siehe Brem, H., Kader, A., Epstein, J.I., Tamargo, R.J., Domb, A. Langer, R. und Leong, K.W. in Sel. Cancer Ther. (1989) 5, 55-65) und Studien mit Ratten (siehe Tamargo, R.J., Epstein, J.I. Reinhard, C.S., Chasin, M. und Brem, H. in J. Biomed. Mater. Res. (1989) 23, 253-266) gezeigt wurde, dass sie für die kontrollierte Freisetzung von Arzneimitteln im Gehirn ohne evidente toxische Wirkungen nützlich sind; xiii) bioabbaubare Polymere, die Orthoestergruppen enthalten, welche für die kontrollierte Freisetzung in vivo eingesetzt wurden (siehe Maa, Y.F. und Heller, J. in J. Control Release (1990) 14, 21-28); und
• xiv) Polyphosphazene, welche anorganische Polymere sind, die aus alternierenden Phosphor- und Stickstoffatomen bestehen (siehe Crommen, J.H., Vandorpe, J. und Schacht, E.H. in J. Control. Release (1993) 24, 167-180).

Other potentially useful polymer spacer materials include:

• i) copolymers of methyl methacrylate with methacrylic acid; these may be erodible (see Lee, PI in Pharm. Res. (1993) 10, 980) and the carboxylate substituents may cause a higher degree of expansion than with neutral polymers;
• ii) block copolymers of polymethacrylates with biodegradable polyesters (see, eg, San Roman, J. and Guillen-Garcia, P. in Biomaterials (1991) 12, 236-241);
• iii) cyanoacrylates, ie polymers of esters of 2-cyanoacrylic acid - these are biodegradable and have been used in the form of nanoparticles for selective drug administration (see Forestier, F., Gerrier, P., Chaumard, C., Quero, AM, Couvreur, P. and Labarre, C. in J. Antimicrob. Chemoter. (1992) 30, 173-179);
• iv) polyvinyl alcohols which are water-soluble and generally considered to be biocompatible (see eg Langer, R. in J. Control, Release (1991) 16, 53-60);
• v) Copolymers of vinyl methyl ether with maleic anhydride found to be bioerodible (see Finne, U., Hannus, M., and Urtti, A., Int. J. Pharm. (1992) 78, 237-241) ;
• vi) polyvinylpyrrolidones, eg having a molecular weight less than about 25,000, which are rapidly filtered by the kidneys (see Hespe, W., Meier, AM and Blankwater, YM in Arzheim. - Research / Drug Res. (1977) 27, 1158-1162);
• vii) Polymers and copolymers of short chain aliphatic hydroxy acids such as glycolic, lactic, butyric, valeric and capric acids (see eg Carli, F. in Chim. Ind. (Milan) (1993) 75, 494-9), including Copolymers that incorporate aromatic hydroxy acids to increase their rate of degradation (see Imasaki, K., Yoshida, M., Fukuzaki, H., Asano, M., Kumakura, M., Mashimo, T., Yamanaka, H., and Nagai T. in Int J J Pharm. (1992) 81, 31-38);
• viii) polyesters consisting of alternating units of ethylene glycol and terephthalic acid, eg Da cron ^R , which are not degradable, but highly biocompatible;
• ix) block copolymers comprising biodegradable segments of aliphatic hydroxy acid polymers (see Younes, H., Nataf, PR, Cohn, D., Appelbaum, YJ, Pizov, G. and Uretzky, G. in Biomater Artif Cells Artif ) 16, 705-719), for example in connection with polyurethanes (see Kobayashi, H., Hyon, SH and Ikada, Y. in "Watercurable and Biodegradable prepolymers" - J. Biomed. Mater. Res. (1991) 25, 1481 -1494);
• x) polyurethanes which are well tolerated in implants and which can be combined with flexible "soft" segments, eg poly (tetramethylene glycol), poly (propylene glycol) or poly (ethylene glycol), and aromatic "hard" segments, eg 4,4'-methylenebis (phenylene isocyanate) (see, for example, Ratner, BD, Johnston, AB and Lenk, TJ in J. Biomed., Mater Res: Applied Biomaterials (1987) 21, 59-90, Sa Da Costa, V in J. Coll., Interface Sci. (1981) 80, 445-452 and Affrossman, S. et al., Clinical Materials (1991) 8, 25-31);
• xi) poly (1,4-dioxan-2-ones) which may be considered biodegradable esters in view of their hydrolyzable ester compounds (see, eg, Song, CX, Cui, XM, and Schindler, A. in Med. Biol. Eng. Comput (1993) 31, S147-150), and may include glycolide units to improve their absorbency (see Bezwada, RS, Shalaby, SW and Newman, HDJ in Agricultural and Synthetic Polymers: Biodegradability and Utilization (1990) (Ed. Glass , JE and Swift, G.), 167-174 - ACS Symposium Series, # 433, Washington DC, USA - American Chemical Society);
• xii) Polyanhydrides, such as copolymers of sebacic acid (octanedioic acid) with bis (4-carboxyphenoxy) propane, for use in studies with rabbits (see Brem, H., Kader, A., Epstein, JI, Tamargo, RJ, Domb, A. Langer, R. and Leong, KW in Sel. Cancer Ther. (1989) 5, 55-65) and rat studies (see Tamargo, RJ, Epstein, JI Reinhard, CS, Chasin, M. and Brem, H. in J. Biomed. Mater. Res. (1989) 23, 253-266) has been shown to be useful for the controlled release of drugs in the brain without evident toxic effects; xiii) biodegradable polymers containing orthoester groups used for controlled release in vivo (see Maa, YF and Heller, J. in J. Control Release (1990) 14, 21-28); and
• xiv) polyphosphazenes which are inorganic polymers consisting of alternating phosphorus and nitrogen atoms (see Crommen, JH, Vandorpe, J. and Schacht, EH in J. Control, Release (1993) 24, 167-180).

The following tables list bonding agents that may be useful in targeting agents in accordance with the invention. Heterobifunctional linking agents connecting means Reactivity 1 Reactivity 2 Remarks ABH carbohydrate Photo reactive ANB-NOS -NH ₂ Photo reactive APDP (1) SH Photo reactive Iodable disulfide linker APG -NH ₂ Photo reactive Reacts selectively with Arg at pH 7-8 ASIB (1) SH Photo reactive Iodierbar ASBA (1) COOH Photo reactive Iodierbar EDC -NH ₂ COOH Zero-length linker GMBS -NH ₂ SH Sulfo-GMBS -NH ₂ SH Water soluble HSAB -NH ₂ Photo reactive Sulfo HSAB -NH ₂ Photo reactive Water soluble MBS -NH ₂ SH Sulfo-MBS -NH ₂ SH Water soluble M ₂ C ₂ H carbohydrate SH MPBH carbohydrate SH NHS-ASA (1) -NH ₂ Photo reactive Iodierbar Sulfo-NHS-ASA (1) -NH ₂ Photo reactive Water soluble, iodable Sulfo-NHS-LC-ASA (1) -NH ₂ Photo reactive Water-soluble, iodizable PDPH carbohydrate SH Disulfidlinker PNP DTP -NH ₂ Photo reactive SADP -NH ₂ Photo reactive Disulfidlinker Sulfo-SADP -NH ₂ Photo reactive Water-soluble disulfide linker SAED -NH ₂ Photo reactive Disulfidlinker SAND -NH ₂ Photo reactive Water-soluble disulfide linker SANPAH -NH ₂ Photo reactive Sulfo SANPAH -NH ₂ Photo reactive Water soluble SASD (1) -NH ₂ Photo reactive Water-soluble iodinatable disulfide linker SIAB -NH ₂ SH Sulfo-SIAB -NH ₂ SH Water soluble SMCC -NH ₂ SH Sulfo-SMCC -NH ₂ SH Water soluble SMPB -NH ₂ SH Sulfo-SMPB -NH ₂ SH Water soluble SMTP -NH ₂ SH Sulfo-LC-SMPT -NH ₂ SH Water soluble SPDP -NH ₂ SH Sulfo-SPDP -NH ₂ SH Water soluble Sulfo-LC-SPDP -NH ₂ SH Water soluble Sulfo-Samca (2) -NH ₂ Photo reactive Sulfo SAPB -NH ₂ Photo reactive Water soluble Notes: (1) = iodable; (2) = fluorescent Homobifunctional bonding agent connecting means Reactivity Remarks BS -NH ₂ BMH SH BASED (1) Photo reactive Iodable disulfide linker BSCOES -NH ₂ Sulfo BSCOES -NH ₂ Water soluble DFDNB -NH ₂ DMA -NH ₂ DMP -NH ₂ DMS -NH ₂ DPDPB SH Disulfidlinker DSG -NH ₂ DSP -NH ₂ Disulfidlinker DSS -NH ₂ DST -NH ₂ Sulfo-DST -NH ₂ Water soluble DTBP -NH ₂ Disulfidlinker DTSSP -NH ₂ Disulfidlinker EGS -NH ₂ Sulfo-EGS -NH ₂ Water soluble SPBP -NH ₂ biotinylation medium Reactivity Remarks Biotin-BMCC SH Biotin-DPPE * Preparation of biotinylated liposomes Biotin-LC-DPPE * Preparation of biotinylated liposomes Biotin HPDP SH Disulfidlinker biotin hydrazide carbohydrate Biotin-LC-hydrazide carbohydrate iodoacetyl-LC-biotin -NH ₂ NHS iminobiotin -NH ₂ Reduced affinity for avidin NHS-SS-Biotin -NH ₂ Disulfidlinker Photoactivatable biotin nucleic acids Sulfo-NHS-biotin -NH ₂ Water soluble Sulfo-NHS-LC-biotin -NH ₂ Notes: DPPE = Dipa imitoyl phosphatidylethanolam in; LC = long chains Means for protein modification medium Reactivity function Ellmann's reagent SH quantified / detects / protects DTT -SS- reduction 2-mercaptoethanol -SS- reduction 2-Mercaptylamin -SS- reduction Traut's reagent -NH ₂ introduces -SH SATA -NH ₂ introduces protected -SH AMCA-NHS -NH ₂ fluorescent marker AMCA hydrazide carbohydrate fluorescent marker AMCA HPDP -SS- fluorescent marker SBF-chloride -SS- Fluorescence detection of -SH N-ethylmaleimide -SS- blocked -SH NHS acetate -NH ₂ blocked and acetylated -NH ₂ citraconic -NH ₂ Blocks reversibly and introduces negative charges DTPA -NH ₂ introduces a chelator BNP's-skatole tryptophan cleaves tryptophan residue Bolton-Huber para-iodophenylalanine -NH ₂ introduces an iodinatable group

In addition to the straight and branched PEG-like linkers already contemplated (eg, polyethylene glycols and others containing from 2 to 100 repeating units of ethylene oxide), linkers in the VLR system may independently be a chemical bond or the residue of a linking group. The term "linking group moiety" as used herein refers to a moiety that remains, results or is obtained from the reaction of a vector reactive group with a reactive site on a vector. The term "vector reactive group" as used herein refers to a belie a group which can react with functional groups typically found on vectors whose derivatization only minimally affects the ability of the vector to bind to its receptor. However, it is especially contemplated that such vector reactive groups can also react with functional groups typically found on relevant protein molecules. Thus, in one aspect, the linkers useful in the practice of this invention are obtained from those groups which can react with any relevant molecules having a vector as described above containing a reactive group, whether such a relevant molecule Protein is or not to form a linking group.

Preferred linking groups are obtained from vector reactive groups selected from, but not limited to:
a group that reacts directly with carboxy, aldehyde, amine (NHR), alcohols, sulfhydryl groups, activated methylenes, and the like on the vector, for example, active halogen-containing groups including, for example, chloromethylphenyl groups and chloroacetyl- [ClCH ₂ C (= O) -] Groups activated 2- (leaving group substituted) ethylsulfonyl and ethylcarbonyl groups such as 2-chloroethylsulfonyl and 2-chloroethylcarbonyl; vinylsulfonyl; vinylcarbonyl; epoxy; Isocyanato, isothiocyanato; Aldehyde; aziridine; succinimidoxycarbonyl; activated acyl groups, such as carboxylic acid halides; mixed Anyhdride and the like.

A group that can readily react with modified vector molecules containing a vector-reactive group, eg, vectors containing a reactive group modified to contain reactive groups such as those mentioned in the tables above, for example, by oxidation of the vector to an aldehyde or a carboxylic acid, in which case the "linking group" of reactive groups selected from amino, alkylamino, arylamino, hydrazino, alkylhadrazino, arylhydrazino, carbazido, semicarbazido, thiocarbazido, thiosemicarbazido, sulfhydryl, sulfhydrylalkyl , Sulfhydrylaryl, hydroxy, carboxy, carboxyalkyl and carboxyaryl. The alkyl moieties of said linking groups can contain from 1 to about 20 carbon atoms. The aryl moieties of said linking groups may contain from about 6 to about 20 carbon atoms; and a group which can be linked to the vector containing a reactive group or to the modified vector as mentioned above by using a crosslinking agent. The residues of certain useful crosslinking agents, such as homobifunctional and heterobifunctional gelatin hardeners, bisepoxides and bisisocyanates can become part of a linking group during the crosslinking reaction. However, other useful crosslinking agents can facilitate crosslinking, for example, such as consumable catalysts, and are not present in the final conjugate. Examples of such crosslinking agents are carbodiimide and carbamoylonium crosslinking agents, as in US 4,421,847 discloses and the ethers of US 4,877,724 , In these crosslinking agents, one of the reactants, such as the vector, must have a carboxyl group and the other, such as a long chain spacer, must have a reactive amine, alcohol or sulfhydryl group. In amide bond formation, the crosslinking agent first reacts selectively with the carboxyl group and is then cleaved off during the reaction of the thus "activated" carboxyl group with an amine to form an amide compound therebetween, thus covalently bonding the two moieties. An advantage of this approach is that the crosslinking of similar molecules, eg, vector to vector, is avoided, whereas the reaction of, for example, homobifunctional crosslinkers is non-selective and undesirable crosslinked molecules are obtained.

Preferred useful linking groups are obtained from various heterobifunctional crosslinking reagents, such as those listed in Pierce Chemical Company Immunotechnology Catalog - Protein Modification Section (1995 and 1996). Useful non-limiting examples of such reagents include:
Sulfo-SMCC sulfosuccinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate
Sulfo-SIAB sulfosuccinimidyl (4-iodoacetyl) aminobenzoate
Sulfo-SMPB sulfosuccinimidyl-4- (p-maleimidophenyl) butyrate
2-IT 2-iminothiolane
SATA N-succinimidyl-S-acetylthioacetate.

In addition to the above description, all or part of the linking groups may also consist of and be obtained from complementary sequences of nucleotides and residues of nucleotides, both naturally occurring and modified, preferably non-self-associating oligonucleotide sequences. Particularly useful, non-limiting reagents for the incorporation of modified nucleotide units containing reactive functional groups, such as amine and sulfhydryl groups, in an oligonucleotide sequence are commercially available from, for example, Clontech Laboratories Inc. (Palo Alto California) and include Uni-Link AminoModifier (Catalog # 5190), biotin-ON phosphoramidite (Catalog # 5191), N-MNT-C6 Amino Modifier (Catalog # 5202), AminoModifier II (Catalog # 5203), DMT-C6-3 Amine-ON (Catalog # 5222), C6 Thiol Modifier (Catalog # 5211), and the like. In one aspect, the linking groups of this invention are obtained from the reaction of a reactive functional group, such as an amine or sulfhydryl group, as available in the Clontech reagents listed above, one or more of which have been incorporated into an oligonucleotide sequence, with, for example, one or more of the vector reactive groups described above, such as a heterobifunctional group on the vector.

By Binding two complementary Oligonucleotide sequences, one to the vector and the other to the reporter, assigns the resulting double-stranded hybridized Oligonucleotide then the linking group between the vector and the reporter.

Other polymer systems that serve as linkers include:
Poly (L or D or DL amino acids) = proteins and peptides; naturally occurring or synthetic
Pseudo-poly (amino acids) = (amino acids linked by non-amide bonds);
Poly (L- or D- or DL-lactide) and the copolymers, eg poly (L-lactide / DL-lactide)
Poly (glycolide)
L-lactide / glycolide copolymers
Poly-caprolactone and its copolymers
polyanhydrides
Poly (orthoester)
polyphosphazenes
Long-chain, straight or branched lipids (& phospholipids)
Sugar and carbohydrates
Oligonucleotides (see above)
as well as mixtures of the above.

Connecting means those according to the invention are used, generally connecting the vector with the reporter or the reporter with the reporter with some Degree of specificity bring, and can also be used to one or more therapeutically effective Means to bind.

The The present invention accordingly provides a tool for therapeutic Drug administration in combination with vector-mediated Direct the product to the desired one Get ready. By "therapeutic" or "medicinal" is meant a means the cheap one Effect on a specific disease in a living human or non-human animal possesses.

therapeutic Connections can inside a molecular aggregate or particulate linker encapsulated or to the encapsulating walls of a vesicular linker bound or incorporated therein. Therefore, the therapeutic Be bound to a part of the surface, for example by covalent or ionic bonds, or may be physical be mixed in an encapsulating material, especially if the drug is a polarity or solubility similar to the material possesses, so as to keep it from the product to escape before it is intended to work in the body. The release of the drug can only by wetting Contact with blood following administration or as a result other internal or external influences, e.g. Resolution processes, which are catalyzed by enzymes, or the use of magnetic Heating, where the reporter is a magnetic particle.

The Therapeutic substance may be covalently attached to the encapsulating membrane surface of a vesicular Linkers are bound using a suitable coupling agent, e.g. as described here. Therefore, you can, for example, initially Phospholipidderivat produce, to which the drug by a biodegradable bond or linker is bound, and then this Derivative in the material used to make the vesicle membrane was installed as described above.

alternative The agent may be initial without the therapeutic agent, which is then added to particulate (e.g. vesicular) Agent is coupled before use or applied to it becomes. Therefore could for example, a therapeutic to a suspension of liposomes in watery Be given medium and shaken to bind the therapeutic to the liposomes or to it to be held liable.

The therapeutic agent may be, for example, a drug or a prodrug suitable for use known to combat angiogenesis or tumors.

By Targeting of an agent that activates a prodrug Contains enzyme, in areas of pathology, one can map the targeting of the enzyme, what is possible makes visualize when the agent finishes the target and if the means of non-target areas disappeared. In this way you can get the optimal time for the injection of the prodrug in individual patients.

A Another alternative is to activate a prodrug, a prodrug Enzyme and a vector in the same particulate linker reporter on a incorporate such a way that the prodrug only after an external Stimulus will be activated. Such a stimulus can be, for example the light stimulation of a chromophore reporter or the magnetic Heating a superparamagnetic reporter after the desired targeting was achieved.

So-called Prodrugs can also in agents according to the invention be used. Therefore, drugs can be derivatized to change their physicochemical properties and to adapt the means of the invention; such derivatized Medicines can are considered to be prodrugs and are usually inactive until one Cleavage of the derivatizing group the active form of the drug regenerated.

therapeutics can be easily delivered to sites of angiogenesis.

To the Example where the reporter is a chelated metal species (for Example, a paramagnetic metal ion or a metal radionuclide), For example, the linker may comprise a chain attached to a metal chelating group is bound, a polymeric chain with a plurality of metal chelating groups attached laterally to the molecular backbone or into the molecular backbone are incorporated, a branched polymer with metal chelating Groups at branch termini (e.g., a dendrimeric polychelant), etc. What for The linker required is simply that he is the vector and Reporters units together for a reasonable amount of time. With reasonable time is meant a period of time sufficient for the contrast agent is to his desired To exert effects e.g. during in vivo contrast to improve a diagnostic imaging procedure.

Therefore It can be under certain circumstances he wishes be that the linker biodegraded after administration becomes. By selecting a suitable biodegradable linker, it is possible to reconcile the biodistribution and bioelimination patterns for to modify the vector and / or reporter. Where the vector and / or the reporters are biologically active or capable of causing adverse effects exercise, if he withheld after the imaging process is over, it may be desirable to a linker biodegradability plan to have a suitable bioelimination or a metabolic Decomposition of the vector and / or reporter units ensures. Therefore For example, a linker may contain a biodegradable function when decomposing Gives decomposition products with modified biodistribution patterns, from the release of the reporter from the vector or from the Fragmentation of a macromolecular structure result. For example is it for Linkers carrying chelated metal ion reporters, possible that the linker has incorporated a biodegradable function that decomposes releases a precipitable chelate compound, which is the reporter contains.

Accordingly, biodegradable Functions, if desired, be incorporated within the linker structure, preferably at sites the (a) branching sites, (b) at or near binding sites for vectors or Reporters, or (c) are such that biodegradation is physiological tolerable or rapidly excreted fragments.

Examples suitable biodegradable functions include ester, amide, Double ester, phosphoester, ether, thioether, guanidyl, acetal and ketal functions.

As discussed above, the linker group, if desired, in its molecular backbone Groups have incorporated the biodistribution of the contrast agent affect or provide a suitable spatial conformation for the contrast agent ensure, e.g. water access to chelated paramagnetic metal ion reporters to enable. For example, the linker backbone partially or substantially all of one or more polyalkylene oxide chains consist.

Thus, the linker can be viewed as a composite of optionally biodegradable vector binding (V _b ) and reporter binding (R _b ) groups linked via linker backbone (L _b ) groups, where the lin Kerf-backbone groups may carry linker side chain (L _sc ) groups to modify biodistribution, etc., and may incorporate biodegradable functions themselves. The R _b and V _b bonding groups may be on the side of the linker backbone or may be on link backbone minima, for example with an R _b or V _b group on a L _b terminus, where R _b or V _b groups are two L _b Connect termini or a L _b termini bears two or more R _b or V _b groups. The L _b and L _SC groups will desirably be oligomeric or polymeric structures (eg, polyesters, polyamides, polyethers, polyamines, oligopeptides, polypeptides, oligo- and polysaccharides, oligonucleotides, etc.), preferably structures which are at least partially hydrophilic or polymeric possess lipophilic nature, such as hydrophilic, amphiphilic or lipophilic structures.

Of the Linker may have a low, medium or high molecular weight own, e.g. up to 2 MD. Generally, linkers are of higher molecular weight if preferred with a variety of vectors or Reporters should be loaded or, if necessary, the vector and the Spatially separating reporters, or if the linker itself serves a role in the modification to offer the biodistribution. In general, however, linkers a molecular weight of 100 to 100,000 D, in particular 120 D to 20 kD own.

The Conjugation of the linker with the vector and the linker with the reporter may be by any suitable chemical conjugation technique accomplished be, e.g. covalent bonding (e.g., ester or amide formation), metal chelation or another metal-coordinative or ionic bond, again as described above.

Examples of suitable linker systems include the magnifier polychelant structures of US-A-5364613 and PCT / EP90 / 00565 , Polyamino acids (eg, polylysine), functionalized PEG, polysaccharides, glycosaminoglycans, dendritic polymers, as in WO93 / 06868 and Tomalia et al. in Angew. Chem. Int. Ed. Engl. 29: 138-175 (1990), PEG chelating polymers as described in U.S. Pat WO94 / 08629 . WO94 / 09056 and WO96 / 26754 described, etc.

Where the reporter is a chelated metal ion, the linker group will generally incorporate the chelant moiety. Alternatively, the chelated metal may be supported on or in a particulate reporter. In any event, conventional metal-chelating groups as known in the fields of radiopharmaceuticals and MRI contrast media may be used, eg, linear, cyclic and branched polyamino-polycarboxylic acids and phosphorus oxygen equivalents, and other sulfur and / or nitrogen ligands. which are known in the art, eg DTPA, DTPA-BMA, EDTA, DO3A, TMT (see for example US-A-5367080 ), BAT and analogs (see, for example, Ohmono et al., J. Med. Chem. 35: 157-162 (1992) and Kung et al., J. Nucl. Med. 25: 326-332 (1984)), N ₂ S ₂ chelator ECD from Neurolite, MAG (see Jurisson et al., Chem. Rev. 93: 1137-1156 (1993)), HIDA, DOXA (1-oxa-4,7,10-triazacyclododecane triacetic acid), NOTA (1 , 4,7-triazacyclononanetriacetic acid), TETA (1,4,8,11-tetraazacyclotetradecane tetraacetic acid), THT 4 '- (3-amino-4-methoxyphenyl) -6,6'-bis (N', N'-dicarboxymethyl- N-methylhydrazino) -2,2 ': 6', 2 "terpyridine), etc. In this regard, the reader is referred to the patent literature of Sterling Winthrop, Nycomed (including Nycomed Imaging and Nycomed Salutar), Schering, Mallinckrodt, Bracco and Squibb, which refer to chelating agents for diagnostic metals, eg in MR, X-ray and radiodiagnostic agents. See for example US-A-4647447 . EP-A-71564 . US-A-4687659 . WO89 / 00557 . US-A-4885363 and EP-A-232751 ,

reporter

The Reporter units in the contrast agents of the invention may have a be any unit that is the definition either direct or indirectly in an in vivo diagnostic imaging procedure are accessible, e.g. Units that emit detectable radiation or can be arranged to emit them (e.g., by radioactive decay, fluorescence excitation, Spin resonance excitation, etc.), units that are local electromagnetic Fields (e.g., paramagnetic, superparamagnetic, ferrimagnetic or ferromagnetic species), units which Absorb or scatter radiant energy (e.g., chromophores and Fluorophores), particles (including fluid-containing vesicles), heavy elements and compounds thereof and units having a generate detectable substance, etc.

A very wide range of materials detectable by diagnostic imaging modalities is known in the art, and the reporter will be selected according to the imaging modality to be used. Thus, for example, for ultrasound imaging, an echogenic material or a material capable of producing an echogenic material will normally be selected; for X-ray imaging, the reporter will generally be or contain a heavy atom (eg for MR imaging, the reporter will either be a non-zero nuclear spin isotope (such as ¹⁹ F) or a material that has unpaired electron spins and therefore paramagnetic, superparamagnetic, ferrimagnetic or ferromagnetic properties for a For light imaging, the reporter will be a light scatterer (eg, a colored or uncolored particle), a light absorber or a light emitter, for magnetometric imaging the reporter will have detectable magnetic properties, for electrical impedance imaging the reporter will affect the electrical impedance and for scintigraphy, SPECT, PET, etc. the reporter will be a radionuclide.

Examples of suitable reporters are well known in the literature of diagnostic imaging, eg, magnetic iron oxide particles, vesicles containing X-ray contrast agents, chelated paramagnetic metals (eg, Gd, Dy, Mn, Fe, etc.). See for example US-A-4647447 . PCT / GB97 / 00067 . US-A-4863715 . US-A-4770183 . WO96 / 09840 . WO85 / 02772 . WO92 / 17212 . PCT / GB97 / 00459 . EP-A-554 213 . US-A-5228446 . WO91 / 15243 . WO93 / 05818 . WO96 / 23524 . WO96 / 17682 . US-A-5387080 . WO95 / 26205 . GB9624918.0 , Etc.

Particularly preferred reporters are: chelated paramagnetic metal ions such as Gd, Dy, Fe and Mn, especially when chelated by macrocyclic chelating groups (eg tetraazacyclododecane chelating agents such as DOTA, DO3A, HP-DO3A and analogs thereof) or by chelating Linker groups such as DTPA, DTPA-BMA, EDTA, DPDP, etc .; ^{Metal radionuclides} such as ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ⁴⁷ Sc, ⁶⁷ / Ga, ⁵¹ Cr, ^177m Sn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁷ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au ²⁰³ Pb, and ¹⁴¹ Ce; superparamagnetic iron oxide crystals; Chromophores and fluorophores with absorption and / or emission maxima in the range from 300 to 1400 nm, in particular 600 nm to 1200 nm, in particular 650 to 1000 nm; chelated heavy metal cluster ions (eg, W or Mo polyoxoanions or the sulfur or mixed oxygen / sulfur analogs); covalently bonded non-metal atoms which either have a high atomic number (eg iodine) or are radioactive, eg ¹²³ I, ¹³¹ I, etc., atoms; iodinated compound-containing vesicles; Etc.

Generally said reporter (1) can be a chelatable metal or a polyatomic metal-containing ion (e.g., TcO, etc.), wherein the metal is a high atomic number metal (e.g., an atomic number greater than 37), a paramagnetic species (e.g., a transition metal or lanthanide), or a radioactive isotope, (2) a covalently bonded non-metallic Species having an unpaired electron site (e.g., a Oxygen or carbon in a stable free radical) Non-metal of a high atomic number or a radioisotope, (3) a polyatomic cluster or crystal containing atoms of high atomic number, and showing cooperative magnetic behavior (e.g., supra-magnetism, Ferrimagnetism or ferromagnetism) or radionuclides, (4) a chromophore (including species with this term are fluorescent or phosphorescent), e.g. a inorganic or organic structure, in particular a complexed Metal ion or an organic group with an extended delocalized Electron system, or (5) a structure or a group with varying Properties of electrical impedance, e.g. due to an extended delocalized electron system.

Examples of particular preferred reporter groups are described in more detail below:
Chelated metal reporters: metal radionuclides, paramagnetic metal ions, fluorescent metal ions, heavy metal ions and cluster ions

Preferred metal radionuclides include ⁹⁰ Y, ^99m Tc, ¹¹¹ In, ⁴⁷ Sc, ⁶⁷ Ga, ⁵¹ Cr, ^177m Sn, ⁶⁷ Cu, ¹⁶⁷ Tm, ⁹⁷ Ru, ¹⁸⁸ Re, ¹⁷⁷ Lu, ¹⁹⁹ Au, ²⁰³ Pb and ¹⁴¹ Ce.

preferred paramagnetic metal ions include ions of transition and lanthanide metals (e.g. Metals with atomic numbers of 6 to 9, 21-29, 42, 43, 44 or 57-71), in particular ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu, in particular of Mn, Cr, Fe, Gd and Dy, more especially Gd.

preferred fluorescent metal ions include lanthanides, especially La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu. Eu is particularly preferred.

Preferred heavy metal-containing reporters may contain atoms of Mo, Bi, Si and W, and in particular they may be polyatomic cluster ions (eg Bi compounds and W and Mo oxides) as in WO91 / 14460 . WO92 / 17215 . WO96 / 40287 and WO96 / 22914 described.

The metal ions are desirably chelated by chelating groups on the linker moiety or in or on a particle (eg, a vesicle or a porous or nonporous inorganic or or ganic solid), in particular linear, macrocyclic, terpyridine and N ₂ S ₂ chelating agents such as DTPA, DTPA-BMA, EDTA, DO3A, TMT. Further examples of suitable chelating groups are in US-A-4647447 . WO89 / 00557 . US-A-5367080 . US-A-5364613 , etc. disclosed.

The Linker unit or the particle may have one or more such Chelating groups contain, if desired, metalated by a or multiple metal species (e.g., to yield reporters present in different Imaging modalities are detectable).

Especially where the metal is not radioactive, it is preferred that a polychelant Left or a particulate Reporters are used.

A chelating or chelating group as mentioned herein the remainder of one or more of a wide variety of chelating Comprise agents that complex a metal ion or a polyatomic ion can (e.g., TcO).

As well known, a chelating agent is a compound, contains the donor atoms, which combine by coordinative bonding with a metal ion can, to form a cyclic structure containing a chelation complex or a chelate is called. This class of compounds is in the Kirk-Othmer Encyclopedia of Chemical Technology, Vol. 5, 339-368 described.

The balance of a suitable chelating agent may be selected from polyphosphates such as sodium tripolyphosphate and hexametaphosphoric acid; Aminocarboxylic acids such as ethylenediaminetetraacetic acid, N- (2-hydroxy) ethylenediaminetriacetic acid, nitrilotriacetic acid, N, N-di (2-hydroxyethyl) glycine, ethylenebis (hydroxyphenylglycine) and diethylenetriaminepentaacetic acid; 1,3-diketones such as acetylacetone, trifluoroacetylacetone and thenoyltrifluoroacetone; Hydroxycarboxylic acids such as tartaric acid, citric acid, gluconic acid and 5-sulfosalicylic acid; Polyamines such as ethylenediamine, diethylenetriamine, triethylenetetraamine and triaminotriethylamine; Amino alcohols such as triethanolamine and N- (2-hydroxyethyl) ethylenediamine, aromatic heterocyclic bases such as 2,2'-diimidazole, picolinamine, dipicolinamine and 1,10-phenanthroline; Phenols such as salicylaldehyde, disulfopyrocatechol and chromotropic acid; Aminophenols such as 8-hydroxyquinoline and oximesulfonic acid, oximes such as dimethylglyoxime and salicylaldoxime; Peptides containing proximal chelating functionality, such as polycysteine, polyhistidine, polyaspartic acid, polyglutaric acid, or combinations of such amino acids; Schiff bases such as disalicylaldehyde-1,2-propylenediimine; Tetrapyrroles such as tetraphenylporphine and phthalocyanine; Sulfur compounds such as toluenedithiol, meso-2,3-dimercaptosuccinic acid, dimercaptopropanol, thioglycolic acid, potassium ethylxanthate, sodium diethyldithiocarbamate, dithizone, diethyldithiophosphoric acid and thiourea; synthetic macrocyclic compounds such as dibenzo [18] crown-6, (CH ₃ ) ₆ - [14] -4,11] -diene-N ₄ and (2,2,2-cryptate), phosphonic acids such as nitrilotrimethylenephosphonic acid, ethylenediaminetetra (methylenephosphonic acid ) and hydroxyethylidenediphosphonic acid or combinations of two or more of the above mentioned agents. The remainder of suitable chelating agents preferably comprise a polycarboxylic acid group and preferred examples include: ethylenediamine-N, N, N ', N'-tetraacetic acid (EDTA); N, N, N ', N ", N" -diethylenetriamine pentaacetic acid (DTPA); 1,4,7,10-tetraazacyclododecane-N, N ', N ", N" -tetraacetic acid (DOTA); 1,4,7,10-tetraazacyclodeodecane-N, N ', N "-triacetic acid (DO3A); 1-oxa-4,7,10-triazacyclododecane-N, N ', N "-triacetic acid (OTTA); trans (1,2) -cyclohexanediethylenetriamine pentaacetic acid (CDTPA).

Other suitable moieties of chelating agents include proteins modified for the chelation of metals such as technetium and rhenium, as described in U.S. Pat US 5,078,985 is described.

Suitable residues of chelating agents may also be obtained from compounds containing N3S and N2S2, such as those described in U.S. Pat US 4444690 . 4670545 ; 4673562 ; 4897255 ; 4965392 ; 4980147 ; 4988496 ; 5021556 and 5075099 are disclosed.

Other suitable residues of chelating agents are in PCT / US91 / 08253 described.

preferred chelating groups are selected from the group consisting of 2-aminomethylpyridine, imino acetic acid, iminodiacetic acid, ethylenediaminetetraacetic (EDTA), diethylenetriamine pentaacetic acid (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), carbonyliminodiacetic acid, Methyleniminoessigsäure, methyleneiminodiacetic, Ethylenthioethyleniminoessigsäure, Ethylenthioethyleniminodiessigsäure, TMT, a terpyridinyl group, a chelating agent with a Terpyridyl group and a carboxymethylamino group, or a salt any of the foregoing acids. Especially preferred chelating groups are DTPA, DTPA-BMA, DPDP, TMT, DOTA and HPDO3A.

Representative chelating groups are also described in US 5559214 A . WO 9526754 . WO 9408624 . WO 9409056 . WO 94/23333 . WO 9408624 . WO 9408629 A1 . WO 9413327 A1 and WO 9412216 A1 ,

method for metalating any of the present chelating agents within the level of expertise. Metals can turn into a chelating Unit incorporated by any of three general methods are: direct incorporation, template synthesis and / or transmetalation. Direct installation is preferred.

Therefore is it desirable that the metal ion readily complexes with the chelating agent is, for example, merely by suspending or mixing one aqueous solution the moiety containing the chelating agent with a metal salt in an aqueous Solution, which preferably has a pH in the range of about 4 to about 11. The salt may be any salt, but preferred is the salt a water-soluble Salt of the metal, such as a halide salt, and more preferred are those Salts so selected that they do not interfere with the binding of the metal ion to the chelating Intermediate means. The unit containing the chelating agent is preferably in an aqueous solution at a pH between approximately 5 and about 9, more preferably pH between about 6 to about 8. The the chelant-containing moiety can be reacted with buffer salts, As citrate, acetate, phosphate and borate are mixed to the optimum pH to create. Preferably, the buffer salts are selected so that do not interfere with the subsequent binding of the metal ion to the chelating one Intermediate means.

at diagnostic imaging preferably includes the vector linker reporter (VLR) construct relationship from metal radionuclide ion to chelating agent used in such diagnostic imaging applications. In preferred embodiments the molar ratio is sufficient of metal ion per chelating agent of about 1: 1000 until about 1: 1.

In radiotherapeutic applications, the VLR preferably contains a ratio of metal radionuclide ion to chelating agent that is effective in such therapeutic applications. In preferred embodiments, the mole ratio of metal ion per chelating agent ranges from about 1: 100 to about 1: 1. The radionuclide can be selected, for example, from radioisotopes of Sc, Fe, Pb, Ga, Y, Bi, Mn, Cu, Cr, Zn, Ge, Mo, Ru, Sn, Sr, Sm, Lu, Sb, W, Re, Po , Ta and Tl. Preferred radionuclides include ⁴⁴ Sc, ⁶⁴ Cu, ⁶⁷ Cu, ²¹² Pb, ⁶⁸ Ga, ⁹⁰ Y, ¹⁵³ Sm, ²¹² Bi, ¹⁸⁶ Re and ¹⁸⁸ Re. Of these, ⁹⁰ Y is particularly preferred. These radioisotopes may be atomic or preferably ionic.

The following isotopes or isotope pairs can be used for both imaging and therapy without having to change the radiolabeling method or chelator: ⁴⁷ Sc ₂₁ ; ¹⁴¹ Ce ₅₈ ; ¹⁸⁸ Re ₇₅ ; ¹⁷⁷ Lu ₇₁ ; ¹⁹⁹ Au ₇₉ ; ⁴⁷ SC ₂₁ ; ¹³¹ I ₅₃ ; ⁶⁷ CU ₂₉ ; ¹³¹ I ₅₃ and ¹²³ I ₅₃ ; ¹⁸⁸ R ₇₅ and ^99m Tc ₄₃ ; ⁹⁰ Y ₃₉ and ⁸⁷ Y ₃₉ ; ⁴⁷ sc ₂₁ and ⁴⁴ sc ₂₁ ; ⁹⁰ Y ₃₉ and ¹²³ I ₅₃ ; ¹⁴⁶ Sm ₆₂ and ¹⁵³ Sm ₆₂ ; and ⁹⁰ Y ₃₉ and ¹¹¹ In ₄₉ Where the linker moiety contains a single chelating agent, the chelant may be attached directly to the vector moiety, eg via one of the metal coordination groups of the chelating agent having an ester, amide, thioester or thioamide bond with an amine moiety. , Thiol or hydroxy group on the vector. Alternatively, the vector and chelant may be directly attached to the chelant backbone via functionality, eg, a CH ₂ -phenyl NCS group attached to a ring carbon of DOTA as suggested as Meares et al. in JACS 110: 6266-6267 (1988), or indirectly via a homo- or heterobifunctional linker, eg, a bisamine, bis-epoxide, diol, diacid, difunctionalized PEG, etc. In this case, the bifunctional linker desirably becomes a chain of 1 to 200 , preferably 3 to 30 atoms between the vector and the chelating moiety.

wobei Ch eine chelatbildende Einheit und Li eine Linkerrückgratkomponente ist, d.h. der Linker besitzt bevorzugt seitliche Chelatbildner, Chelatbildner im Rückgrat oder terminale Chelatbildner oder eine Kombination davon. Die seitlichen und im Rückgrat befindlichen polymeren Strukturen können verzweigt sein oder sind bevorzugter linear und die Wiederholungseinheiten (LiCh) oder andere Wiederholungseinheiten im Polymer können im Rückgrat befindliche oder seitliche biodistributionsmodifizierende Gruppen besitzen, z.B. Polyalkylengruppen, wie in WO94/08629 , WO94/09056 und WO96/20754 . Die terminalen chelatbildenden Strukturen Li(Ch)_n, die dendritische Polymere wie in WO93/06868 sein können, können biodistributionsmodifizierende Gruppen besitzen, die an Termini gebunden sind, die nicht von Chelatbildnern besetzt sind und können den Bioabbau verbessernde Stellen innerhalb der Linkerstruktur besitzen, wie in WO95/28966 .Where the linker moiety contains a plurality of chelating groups, the linker is or is preferably part of the formulas

wherein Ch is a chelating moiety and Li is a linker backbone component, ie, the linker preferably has side chelants, backbone chelators or terminal chelants, or a combination thereof. The lateral and backbone polymeric structures may be branched or more preferably linear and the repeat units (LiCh) or other repeating units in the polymer may have backbone or lateral biodistribution modifying groups, eg polyalkylene groups as in WO94 / 08629 . WO94 / 09056 and WO96 / 20754 , The terminal chelating structures Li (Ch) _n , the dendritic polymers as in WO93 / 06868 may have biodistribution-modifying groups attached to termini that are not occupied by chelators, and may possess biodegradation sites within the linker structure, as in WO95 / 28966 ,

The chelant moieties within the polychelant-forming linker can be attached via backbone functionalization of the chelant or by utilizing one or more of the metal coordinating groups of the chelant or by amide or ether bond formation between the acid chelant and an amine or hydroxyl-bearing linker backbone, eg, as in polylysine. polyDTPA, polylysine-polyDOTA and in the so-called Magnifierpolychelatbildnern of PCT / EP96 / 00565 , Such polychelant-forming linkers can be conjugated to one or more vector groups either directly (eg, using amine, acid or hydroxyl groups in the polychelant-forming linker) or via a bifunctional linker compound as discussed above for monochelant-forming linkers.

For example, where the chelated species is carried by a particulate (or molecular aggregate, eg vesicular) linker, the chelate may be an unbound mono- or polychelate (such as Gd DTPA-BMA or Gd HP-DO3A) trapped within the particle it may be a mono- or polychelate conjugated to the particle either by covalent bonding or by interaction of an anchor group (eg, a lipophilic group) on the mono / polychelate with the membrane of a vesicle (see, eg, US Pat PCT / GB95 / 02378 ).

Non-metal atomic reporter

Preferred non-metal atomic reporters include radioisotopes such as ¹²³ I and ¹³¹ I as well as non-zero nuclear spin atoms such as ¹⁹ F and heavy atoms such as I.

such Reporter, preferably a plurality thereof, e.g. 2 to 200, can be covalent to a linker backbone be bound, either directly using conventional chemical synthesis techniques or via a support group, e.g. a triiodophenyl group.

In an embodiment This invention will be particularly useful in the use of radioisotopes of iodine Considered. For example, if the vector or linker off Substituents formed by iodine in a covalent bond forming reaction can be chemically substituted, such as substituents containing a hydrophenyl functionality, can such substituents by methods well-known in the art are known to be labeled with a radioisotope of iodine. The Iodine species can in therapeutic and diagnostic imaging applications be used. While at the same time a metal in a chelating agent the same vector linker also in either therapeutic or diagnostic Image forming applications can be used.

As with the metal chelators discussed above, such metal atomic reporters may be attached to the linker or carried in or on a particulate linker, eg, in a vesicle (see WO95 / 26205 and GB9624918.0 ).

left of the type described above in connection with the metal reporters can for non-metal atom reporters using the non-metal atom reporter or groups, which carry such reporters, the site of one or all chelating Take groups.

Organic chromophores or fluorophore reporter

Preferred organic chromophoric and fluorophoric reporters include extended delocalized electron systems such as cyanines, merocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrylium dyes, thiapyrylium dyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones, naphthoquinones, indathrene, phthaloylacridones , Trisphenoquinones, azo dyes, intramolecular and intermolecular charge transfer dyes and dye complexes, tropones, tetrazines, bis (dithiols) complexes, bis (benzoldithiolate) complexes, iodaniline dyes, bis (S, O-dithiols) complexes, etc. Examples of suitable organic or metalated organic chromophores can be found in "Topics in Applied Chemistry: Infrared-absorbing Dyes" ed. M. Matsuoka, Plenum, NY 1990, "Topics in Applied Chemistry: The Chemistry and Application of Dyes", Waring et al. Plenum, NY, 1990, "Handbook of Fluorescent Probes and Research Chemicals" Haugland, Molecular Probes Inc., 1996, DE-A-4445065 . DE-A-4326466 . JP-A-3/228046 , Narayanan et al. J. Org. Chem. 60: 2391-2395 (1995), Lipowska et al. hetero cyclic comm. 1: 427-430 (1995), Fabian et al. Chem. Rev. 92: 1197 (1992), WO96 / 23525 , Strekowska et al. J. Org. Chem. 57: 4578-4580 (1992), WO (Axis) and WO96 / 17628 , Specific examples of chromophores that can be used include xylene cyanol, fluorescein, dansyl, NBD, indocyanine green, DODCI, DTDCI, DOTCI and DDTCI.

Especially preferred are groups, the absorption maxima between 600 and 1000 nm to avoid interference with hemoglobin absorption (e.g., xylene cyanols).

• 4a: wobei Y=S, X=I, R=Et
• 4b: wobei Y=S, X=ClO₄, R=Et
• 4c: wobei Y=CMe₂, X=1, R=Me
• 4d: wobei Y=CMe₂, X=ClO₄, R=Me
• 4e: wobei Y=CH=CH, X=I, R=Et
• 4f: wobei Y=CH=CH, X=Br, R=Et
• 4g: wobei Y=CH=CH, X=ClO₄, R=Et

und in Tabelle III auf Seite 28 von Matsuoka (supra), d.h.

wobei Y=O, X=1, R=Me
wobei Y=CMe₂, X=I, R=Me
wobei Y=S, X=Br, R=Et
Chalcogenpyrylomethinfarbstoffe, z.B. Verbindungen 12 auf Seite 31 von Matsuoka (supra), d.h.

wobei Y = Te, Se, O or NR;
Monochalcogenpyrylomethinfarbstoffe, z.B. Verbindungen 13 auf Seite 31 von Matsuoka (supra), d.h.

wobei n = 1 oder 2;
Pyryliumfarbstoffe, z.B. Verbindungen 14 (X=O) auf Seite 32 von Matsuoka (supra), d.h.

wobei X = O, S oder Se;
Thiapyryliumfarbstoffe, z.B. Verbindungen 15 auf Seite 32 und Verbindung I auf Seite 167 von Matsuoka (supra), d.h.

wobei n = 1 oder 2;
Squaryliumfarbstoffe, z.B. Verbindung 10 und Tabelle IV auf Seite 30 von Matsuoka (supra), d.h.

wobei X = CH=CH, Y = H und R = Et,
X=S, Y=H und R=Et und
X = CMe₂, Y = H und R = Me,
und Verbindung 6, Seite 26 von Matsuoka (supra), d.h.

wobei X = CH=CH, Y = H und R=Et;
Croconiumfarbstoffe, z.B. Verbindung 9 und Tabelle IV auf Seite 30 von Matsuoka (supra), d.h.

wobei X = CH=CH, Y = H und R = Et,
X = S, Y = H und R = Et
X = CMe₂, Y = H und R = Me
und Verbindung 7, Seite 26 von Matsuoka (supra), d.h.

wobei X = CH=CH, Y = H und R = Et;
Azuleniumfarbstoffe, d.h. Verbindung 8 auf Seite 27 von Matsuoka (supra), d.h.

Merocyaninfarbstoffe, z.B. Verbindung 16, R = Me, auf Seite 32 von Matsuoka (supra), d.h.

Indoanilinfarbstoffe, wie Kupfer- und Nickelkomplexe von Indoanilinfarbstoffen, z.B. Verbindung 6 auf Seite 63 von Matsuoka (supra), d.h.

wobei R = Et, R = Me, M = Cu,
R = Et, R' = Me, M = Ni
R = Me, R' = H, M = Cu oder
R = Me, R' = H, M = Ni,
Benzo[a]phenoxaziniumfarbstoffe und Benzo[a]phenothiaziniumfarbstoffe, z.B. wie auf Seite 201 von Matsuoka (supra) gezeigt, d.h.

wobei X = O oder S;
1,4-Diaminoanthrachinon(N-alkyl)-3'-thioxo-2,3-dicarboximide, z.B. Verbindung 20 auf Seite 41 von Matsuoka (supra)

Indanthrenpigmente, z.B.

siehe Verbindung 21 auf Seite 41 von Matsuoka (supra);
2-Arylamino-3,4-phthaloylacridonfarbstoffe, z.B. Verbindung 22 auf Seite 41 von Matsuoka (supra)

Trisphenochinonfarbstoffe, z.B. Verbindung 23 auf Seite 41 von Matsuoka (supra)

Azofarbstoffe, z.B. der Monoazofarbstoff, Verbindung 2 auf Seite 90 von Matsuoka (supra), d.h.

wobei X = CH=C(CN)₂, R₁ = R₂ = Et, R₃ = R₄ = H,
X = C(CN)=C(CN)₂, R₁ = R₂ = Et, R₃ = R₄ = H oder
X=

und Y = C=O, R₁ = R₂ = Et, R₃ = R₄ = H,
oder Y = SO₂, R₁ = H, R₂ = CH (Me) nBu, R₃ = OMe und R₄ = NHAc;
Azofarbstoffe, z.B. der Polyazofarbstoff, Verbindung 5 auf Seite 91 von Matsuoka (supra), d.h.

Intramolekular-Charge-Transfer-Donor-Akzeptor-Infrarotfarbstoffe, z.B. Verbindungen 6 und 7 auf Seite 91 von Matsuoka (supra), d.h.

und

nichtbenzoide aromatische Farbstoffe, z.B. Verbindung 8, ein Tropon, auf Seite 92 von Matsuoka (supra), d.h.

Tetrazinradikalfarbstoffe, z.B. Verbindung 9 auf Seite 92 von Matsuoka (supra), d.h.

worin, X = p-Phenylen oder
X = p-Terphenylen, wie auch Verbindung 10 auf Seite 92 von Matsuoka (supra), d.h.

worin X = p-Biphenyl;
kationische Salze von Tetrazinradikalfarbstoffen, z.B. Verbindung 11 auf Seite 92 von Matsuoka (supra)

worin X = p-Phenylen;
Donor-Akzeptor-Intramolekular-Charge-Transfer-Farbstoffe, z.B. CT-Komplexe von Verbindungen 13b und 14a bis 14c auf Seite 93 von Matsuoka (supra), d.h.

wobei X = CH=N-N(Ph)₂ im Donor und

• a) Y = CN, Z = NO₂
• b) Y = CN, Z = H oder
• c) Y = Cl, Z = NO₂ in dem Akzeptor;

Anthrachinonfarbstoffe, z.B. Verbindungen 12 (X = S oder Se) auf Seite 38 von Matsuoka (supra), d.h.

worin X = S oder Se und Y = Tetrachlor, Tetrabrom, 2,3-Dicarbonsäure, 2,3-Dicarbonsäureanhydrid oder 2,3-Dicarbonsäure-N-phenylimid;
Naphthochinonfarbstoffe, z.B. Verbindungen 2, 3 und 4 auf Seite 37 von Matsuoka (supra), d.h.

und

metallierte Azofarbstoffe, wie Azofarbstoffe, die Nickel, Kobalt, Kupfer, Eisen und Mangan enthalten; Phthalocyaninfarbstoffe, z.B. Verbindung 1 in Tabelle II auf Seite 51 von Matsuoka (supra), z.B.

Naphthalocyaninfarbstoffe, z.B. Verbindung 3 in Tabelle II auf Seite 51 von Matsuoka (supra), z.B.

Metallphthalocyanine, wie Phthalocyanine, die Aluminium, Silicium, Nickel, Zink, Blei, Cadmium, Magnesium, Vanadium, Kobalt, Kupfer und Eisen enthalten, z.B. Verbindung 1 in Tabelle III auf Seite 52 von Matsuoka (supra), z.B.

worin beispielsweise M = Mg;
Metallnaphthocyanine, wie Naphthalocyanine, die Aluminium, Zink, Kobalt, Magnesium, Cadmium, Silicium, Nickel, Vanadium, Blei, Kupfer und Eisen enthalten, siehe Verbindung 3 in Tabelle III auf Seite 52 von Matsuoka (supra), z.B.

worin beispielsweise M = Mg;
Bis(dithiolen)metallkomplexe, die ein Metallion wie Nickel, Kobalt, Kupfer und Eisen koordiniert an vier Schwefelatome in einem Bis(S,S'-bidentat)ligandkomplex aufweisen, z.B. siehe Tabelle I auf Seite 59 von Matsuoka (supra)

wobei
R₁ = R₂ = CF₃, M = Ni
R₁ = R₂ = Phenyl, M = Pd,
R₁ = R₂ = Phenyl, M = Pt,
R₁ = C4 bis C10 Alkyl, R₂ = H, M = Ni,
R₁ = C4 bis C10 Alkyl, R₂ = H, M = Pd,
R₁ = C4 bis C10 Alkyl, R₂ = H, M = Pt,
R₁ = R₂ = Phenyl, M = Ni,
R₁ = R₂ = p-CH₃-Phenyl, M = Ni,
R₁ = R₂ = p-CH₃O-Phenyl, M = Ni,
R₁ = R₂ = p-Cl-Phenyl, M = Ni,
R₁ = R₂ = p-CF₃-Phenyl, M = Ni,
R₁ = R₂ = 3,4-diCl-Phenyl, M = Ni,
R₁ = R₂ = o-Cl-Phenyl, M = Ni,
R₁ = R₂ = o-Br-Phenyl, M = Ni,
R₁ = R₂ = 3,4-diCl-Phenyl, M = Ni,
R₁ = R₂ = p-CH₃, M = Ni,
R₁ = R₂ = 2-Thienyl, M = Ni,
R₁ = p-(CH₃)₂ N-Phenyl, R2 = Phenyl, M = Ni, und
R₁ = p-(CH₃)₂ N-Phenyl, R2 = p-H₂N-Phenyl, M = Ni;
Bis(benzoldithiolat)metallkomplexe, die ein Metallion aufweisen, wie Nickel, Kobalt, Kupfer und Eisen, koordiniert an vier Schwefelatome in einem Ligandkomplex, z.B. siehe Tabelle III auf Seite 62 von Matsuoka (supra), d.h.

wobei
X = Tetramethyl, M = Ni,
X = 4,5-Dimethyl, M = Ni,
X = 4-Methyl, M = Ni,
X = Tetrachlor, M = Ni,
X = H, M = Ni,
X = 4-Methyl, M = Co,
X = 4-Methyl, M = Cu, und
X = 4-Methyl, M = Fe;
N,O-Bidentatindoanilinfarbstoffe, die ein Metallion aufweisen, wie Nickel, Kobalt, Kupfer und Eisen koordiniert an zwei Stickstoff- und zwei Sauerstoffatome von zwei N,O-Bidentatindoanilinliganden, z.B. Verbindung 6 in Tabelle IV auf Seite 63 von Matsuoka (supra), z.B.

wobei R = Et, R' = Me, M = Cu,
R = Et, R' = Me, M = Ni,
R = Me, R' = H, M = Cu, und
R = Me, R' = H, M = Ni,
bis(S,O-Dithiolen)metallkomplexe, die ein Metallion aufweisen, wie Nickel, Kobalt, Kupfer und Eisen koordiniert an zwei Schwefelatome und zwei Sauerstoffatome in einem Bis(S,O-bidentat)ligandkomplex, z.B. siehe US 3,806,462 , z.B.

a-Diimindithiolenkomplexe, die ein Metallion aufweisen, wie Nickel, Kobalt, Kupfer und Eisen koordiniert an zwei Schwefelatome und zwei Imino-Stickstoffatome in einem gemischten S,S- und N,N-Bidentatdiligandkomplex, z.B. siehe Tabelle II auf Seite 180, zweite von unten, von Matsuoka (supra) (siehe auch JP-Patente: 62/39,682 , 63/126,889 und 63/139,303 ), z.B.

und
tris-(a-Diimin)komplexe, die ein Metallion aufweisen, das mit sechs Stickstoffatomen in einem Triligandkomplex koordiniert ist, z.B. siehe Tabelle II auf Seite 180 von Matsuoka (supra), letzte Verbindung (siehe auch JP-Patente 61/20,002 und 61/73,902 ), z.B.

Other such examples include: cyanine dyes such as heptamethine cyanine dyes, eg compounds 4a to 4g Table II on page 26 of Matsuoka (supra)

• 4a: where Y = S, X = I, R = Et
• 4b: where Y = S, X = ClO ₄ , R = Et
• 4c: where Y = CMe ₂ , X = 1, R = Me
• 4d: where Y = CMe ₂ , X = ClO ₄ , R = Me
• 4e: where Y = CH = CH, X = I, R = Et
• 4f: where Y = CH = CH, X = Br, R = Et
• 4g: where Y = CH = CH, X = ClO ₄ , R = Et

and in Table III on page 28 of Matsuoka (supra), ie

where Y = O, X = 1, R = Me
where Y = CMe ₂ , X = I, R = Me
where Y = S, X = Br, R = Et
Chalcogenpyrylomethine dyes, eg compounds 12 on page 31 of Matsuoka (supra), ie

where Y = Te, Se, O or NR;
Monochalcogenpyrylomethine dyes, eg compounds 13 on page 31 of Matsuoka (supra), ie

where n = 1 or 2;
Pyrylium dyes, eg compounds 14 (X = O) on page 32 of Matsuoka (supra), ie

where X = O, S or Se;
Thiapyrylium dyes, eg compounds 15 on page 32 and compound I on page 167 of Matsuoka (supra), ie

where n = 1 or 2;
Squarylium dyes, eg Compound 10 and Table IV on page 30 of Matsuoka (supra), ie

where X = CH = CH, Y = H and R = Et,
X = S, Y = H and R = Et and
X = CMe ₂ , Y = H and R = Me,
and Compound 6, page 26 of Matsuoka (supra), ie

where X = CH = CH, Y = H and R = Et;
Croconium dyes, eg Compound 9 and Table IV on page 30 of Matsuoka (supra), ie

where X = CH = CH, Y = H and R = Et,
X = S, Y = H and R = Et
X = CMe ₂ , Y = H and R = Me
and Compound 7, page 26 of Matsuoka (supra), ie

where X = CH = CH, Y = H and R = Et;
Azulenium dyes, ie compound 8 on page 27 of Matsuoka (supra), ie

Merocyanine dyes, eg Compound 16, R = Me, on page 32 of Matsuoka (supra), ie

Indoaniline dyes, such as copper and nickel complexes of indoaniline dyes, eg Compound 6 on page 63 of Matsuoka (supra), ie

where R = Et, R = Me, M = Cu,
R = Et, R '= Me, M = Ni
R = Me, R '= H, M = Cu or
R = Me, R '= H, M = Ni,
Benzo [a] phenoxazinium dyes and benzo [a] phenothiazinium dyes, eg as shown on page 201 of Matsuoka (supra), ie

where X = O or S;
1,4-diaminoanthraquinone (N-alkyl) -3'-thioxo-2,3-dicarboximides, eg Compound 20 on page 41 of Matsuoka (supra)

Indanthrene pigments, eg

see compound 21 on page 41 of Matsuoka (supra);
2-arylamino-3,4-phthaloylacridone dyes, eg Compound 22 on page 41 of Matsuoka (supra)

Trisphenoquinone dyes, eg Compound 23 on page 41 of Matsuoka (supra)

Azo dyes, for example the monoazo dye, Compound 2 on page 90 of Matsuoka (supra), ie

where X = CH = C (CN) ₂ , R ₁ = R ₂ = Et, R ₃ = R ₄ = H,
X = C (CN) = C (CN) ₂ , R ₁ = R ₂ = Et, R ₃ = R ₄ = H or
X =

and Y = C = O, R ₁ = R ₂ = Et, R ₃ = R ₄ = H,
or Y = SO ₂ , R ₁ = H, R ₂ = CH (Me) n Bu, R ₃ = OMe and R ₄ = NHAc;
Azo dyes, eg the polyazo dye, Compound 5 on page 91 of Matsuoka (supra), ie

Intramolecular charge-transfer donor-acceptor infrared dyes, eg compounds 6 and 7 on page 91 of Matsuoka (supra), ie

and

non-benzoic aromatic dyes, eg Compound 8, a tropon, at page 92 of Matsuoka (supra), ie

Tetrazinradikalfarbstoffe, eg, compound 9 on page 92 of Matsuoka (supra), ie

wherein, X = p-phenylene or
X = p-terphenylene, as well as compound 10 on page 92 of Matsuoka (supra), ie

wherein X = p-biphenyl;
cationic salts of tetrazine radical dyes, eg Compound 11 on page 92 of Matsuoka (supra)

wherein X = p-phenylene;
Donor Acceptor Intramolecular Charge Transfer Dyes, eg CT Complexes of Compounds 13b and 14a to 14c on page 93 of Matsuoka (supra), ie

where X = CH = NN (Ph) ₂ in the donor and

• a) Y = CN, Z = NO ₂
• b) Y = CN, Z = H or
• c) Y = Cl, Z = NO ₂ in the acceptor;

Anthraquinone dyes, eg compounds 12 (X = S or Se) on page 38 of Matsuoka (supra), ie

wherein X = S or Se and Y = tetrachloro, tetrabromo, 2,3-dicarboxylic acid, 2,3-dicarboxylic anhydride or 2,3-dicarboxylic acid N-phenylimide;
Naphthoquinone dyes, eg compounds 2, 3 and 4 on page 37 of Matsuoka (supra), ie

and

metallated azo dyes, such as azo dyes containing nickel, cobalt, copper, iron and manganese; Phthalocyanine dyes, eg Compound 1 in Table II on page 51 of Matsuoka (supra), eg

Naphthalocyanine dyes, eg Compound 3 in Table II on page 51 of Matsuoka (supra), eg

Metal phthalocyanines such as phthalocyanines containing aluminum, silicon, nickel, zinc, lead, cadmium, magnesium, vanadium, cobalt, copper and iron, eg Compound 1 in Table III on page 52 of Matsuoka (supra), eg

wherein, for example, M = Mg;
Metal naphthocyanines such as naphthalocyanines containing aluminum, zinc, cobalt, magnesium, cadmium, silicon, nickel, vanadium, lead, copper and iron, see compound 3 in Table III on page 52 of Matsuoka (supra), eg

wherein, for example, M = Mg;
Bis (dithiolene) metal complexes having a metal ion such as nickel, cobalt, copper and iron coordinated to four sulfur atoms in a bis (S, S'-bidentate) ligand complex, eg see Table I on page 59 of Matsuoka (supra)

in which
R ₁ = R ₂ = CF ₃ , M = Ni
R ₁ = R ₂ = phenyl, M = Pd,
R ₁ = R ₂ = phenyl, M = Pt,
R ₁ = C ₄ to C 10 alkyl, R ₂ = H, M = Ni,
R ₁ = C ₄ to C 10 alkyl, R ₂ = H, M = Pd,
R ₁ = C ₄ to C 10 alkyl, R ₂ = H, M = Pt,
R ₁ = R ₂ = phenyl, M = Ni,
R ₁ = R ₂ = p-CH ₃ -phenyl, M = Ni,
R ₁ = R ₂ = p-CH ₃ O-phenyl, M = Ni,
R ₁ = R ₂ = p-Cl-phenyl, M = Ni,
R ₁ = R ₂ = p-CF ₃ -phenyl, M = Ni,
R ₁ = R ₂ = 3,4-diCl-phenyl, M = Ni,
R ₁ = R ₂ = o-Cl-phenyl, M = Ni,
R ₁ = R ₂ = o-Br-phenyl, M = Ni,
R ₁ = R ₂ = 3,4-diCl-phenyl, M = Ni,
R ₁ = R ₂ = p-CH ₃ , M = Ni,
R ₁ = R ₂ = 2-thienyl, M = Ni,
R ₁ = p- (CH ₃ ) ₂ N-phenyl, R ₂ = phenyl, M = Ni, and
R ₁ = p- (CH ₃ ) ₂ N -phenyl, R ₂ = pH ₂ N-phenyl, M = Ni;
Bis (benzenedithiolate) metal complexes having a metal ion, such as nickel, cobalt, copper and iron, coordinated to four sulfur atoms in a ligand complex, eg see Table III on page 62 of Matsuoka (supra), ie

in which
X = tetramethyl, M = Ni,
X = 4,5-dimethyl, M = Ni,
X = 4-methyl, M = Ni,
X = tetrachloro, M = Ni,
X = H, M = Ni,
X = 4-methyl, M = Co,
X = 4-methyl, M = Cu, and
X = 4-methyl, M = Fe;
N, O-bidentate indoaniline dyes having a metal ion such as nickel, cobalt, copper and iron coordinated to two nitrogen and two oxygen atoms of two N, O-bidentate indoaniline ligands, eg Compound 6 in Table IV on page 63 of Matsuoka (supra), eg

where R = Et, R '= Me, M = Cu,
R = Et, R '= Me, M = Ni,
R = Me, R '= H, M = Cu, and
R = Me, R '= H, M = Ni,
bis (S, O-dithiols) metal complexes having a metal ion such as nickel, cobalt, copper and iron coordinated to two sulfur atoms and two oxygen atoms in a bis (S, O-bidentate) ligand complex, eg see US 3,806,462 , eg

a-Diimindithiolenkomplexe having a metal ion, such as nickel, cobalt, copper and iron coordinated to two sulfur atoms and two imino nitrogen atoms in a mixed S, S and N, N-Bidentatdiligandkomplex, eg see Table II on page 180, second of below, from Matsuoka (supra) (see also JP Patents: 62 / 39,682 . 63 / 126.889 and 63 / 139.303 ), eg

and
tris (a-diimine) complexes having a metal ion coordinated to six nitrogen atoms in a triligand complex, eg, see Table II on page 180 of Matsuoka (supra), final compound (see also Japanese Patent 61 / 20,002 and 61 / 73.902 ), eg

Representative examples of visible dyes include fluorescein derivatives, rhodamine derivatives, coumarins, azo dyes, metalatable dyes, anthraquinone dyes, benzodifuranone dyes, poly cyclic aromatic carbonyl dyes, indigoid dyes, polymethine dyes, azacarbocyanine dyes, hemicyanine dyes, barbiturates, diazahemicyanine dyes, styryl dyes, diaryl carbonium dyes, triaryl carbonium dyes, phthalocyanine dyes, quinophthalone dyes, triphenodioxazine dyes, formazan dyes, phenothiazine dyes such as methylene blue, azure A, azure B and azure C, oxazine dyes, thiazine dyes, naptholactam dyes , Diazahemicyanine dyes, azopyridone dyes, azobenzene dyes, mordant dyes, acid dyes, basic dyes, metallized and premetallized dyes, xanthene dyes, direct dyes, leuco dyes that can be oxidized to form dyes that are bathochromically shifted from those of the precursor leuco dyes, and others Dyes, such as those listed by Waring, DR and Hallas, G. in "The Chemistry and Application of Dyes", Topcis in Applied Chemist Additional dyes can be found listed in Haugland, RP, "Handbook of Fluoride Probes and Research Chemicals", 6th Edition, Molecular Probes, Inc., Eugene OR, 1996.

such Chromophores and fluorophores can either directly to the vector or within a linker structure be covalently bound. Again you can Linker of the type described above together with the metal reporters for organic Chromophores or fluorophores are used, with the choromophore / fluorophores take the place of some or all chelating groups.

As Chromophors / fluorophores can be used with the metal chelators discussed above in or on particulate Linker units are worn, e.g. in or on a vesicle, or can covalently bound to inert matrix particles, also called a Light scattering reporters can serve.

Particulate reporter or linker reporter

The particulate Reporters and the linker reporters generally fall into two categories - those where the particle has a matrix or shell containing the Reporter carries or contains, and those where the particle matrix itself is the reporter. Examples of the first category are: vesicles (e.g., micelles and liposomes), the one liquid or solid phase containing the contrast-effective reporter, e.g. a chelated paramagnetic metal or radionuclide, or a water-soluble iodinated X-ray contrast agent; porous Particles loaded with the reporter, e.g. with paramagnetic Metal-loaded particles of a molecular sieve; and solid particles, e.g. an inert biotolerable polymer to which the reporter is bound or applied, e.g. dye particles loaded with polymer.

Examples the second category are: light-scattering organic or inorganic Particles, magnetic particles (e.g., superparamagnetic, ferromagnetic or ferrimagnetic particles); and dye particles.

Preferred particulate reporters or reporter linkers include superparamagnetic particles (see US-A-4770183 . PCT / GB97 / 00067 . WO96 / 09840 , etc.), echogenic vesicles (see WO92 / 17212 . PCT / GB97 / 00495 , etc.), iodine-containing vesicles (see WO95 / 26205 and GB9624918.0 ) and dye-loaded polymer particles (see WO96 / 23524 ).

The particulate reporters may have one or more vectors that are directly or indirectly bound to their surfaces. In general, it will be preferable to bind a plurality (eg, 2 to 50) of vector units per particle. It is particularly advantageous, in addition to the desired targeting vector, also bind flux-slowing vectors to the particles, ie vectors which have an affinity for Kapillarlumen or other organ surfaces, which is sufficient to reduce the passage of the contrast agent through the capillaries or the target organ, but not sufficient to immobilize the contrast agent. Such flux-slowing vectors (eg described in GB9700699.3 ) may also serve to anchor the contrast agent once bound to its target site.

The Means by which vector-to-particle binding is achieved will depend on the nature of the particle surface. For inorganic particles can the connection with the particle, for example by means of an interaction between a metal linking group (e.g., a phosphate, phosphonate, or oligo- or polyphosphate group) on the vector or on one Linker bound to the vector. For organic (e.g., polymeric) particles can be used to direct vector binding covalent bond between groups on the particle surface and reactive groups in the vector, e.g. Amide or ester bond, or by covalent attachment of the vector and the particle to a linker accomplished become. Linker of the above together with chelated metal reporters of the type discussed although generally the linker is not used be used to couple particles together.

For non-solid particles, eg droplets (eg of water-insoluble iodinated liquids, as in US-A-5318767 . US-A-5451393 . US-A-5352459 and US-A-5569448 described) and vesicles, the linker may desirably contain hydrophobic "anchor" groups, eg saturated or unsaturated C _12-30 chains, which penetrate the particle surface and bind the vector to the particle. Thus, for phospholipid vesicles, the linker may serve to covalently attach the vector to phospholipid compatible with the vesicle membrane. Examples of linker bonding to vesicles and inorganic particles are in GB9622368.0 and PCT / GB97 / 00067 described.

Other than the vectors, other groups can be attached to the particle surface, eg, stabilizers (to prevent aggregation) and biodistribution modifiers, such as PEG. Such groups are for example in PCT / GB97 / 00067 . WO96 / 09840 . EP-A-284549 and US-A-4904479 discussed.

Preferably, the VLR agents of the invention will have the receptor targeting vectors coupled directly or indirectly to a reporter, eg with covalently bound iodine radioisotopes, with metal chelates bound directly or via an organic linker group, or coupled to a particulate reporter or linker reporter, eg superparamagnetic crystals (optionally coated, eg as in PCT / GB97 / 00067 ) or a vesicle, eg a micelle or a liposome containing an iodinated contrast agent.

In short, for the imaging modalities MRI, X-ray, light imaging, nuclear imaging, magnetotomeography, and electrical impedance tomography, the favorite reporters may be: MRI superparamagnetic iron oxide particles, generally having a particle size less than about 80 nm. In particular, iron oxides covered with various covering materials, such as polyelectrolytes, PEG, starch and hydrolyzed starch are preferred. Paramagnetic metal substances including both chelates and particulate materials are also useful. Light imaging Any photo-generating reporter group. The focus should be on substances that absorb near the near infrared. nuclear medicine Radioactive chelates that have ⁹⁹ Tc or ¹¹¹ In, as well as directly radiolabelled vectors with radiolabelled halogen substituents such as ¹²³ I, ¹²⁵ I, ¹³¹ I, ⁷⁵ Br, or ⁷⁷ Br. magnetotomography Superparamagnetic iron oxide particles as described above. Electrical Impedance Tomography Polyionic species, eg polymers with tomography ionic groups in the repeat units.

The Means of the invention can administered to patients for imaging purposes in amounts sufficient around the desired Contrast with the particular imaging technique. Where The reporter is a metal, are generally dosages of approximately 0.001 to 5.0 mmoles of chelated imaging metal ion per kilogram of the patient's body weight effective to obtain adequate contrast enhancements. For the most MRI applications become the preferred dosages of imaging metal ion in the range of 0.02 to 1.2 mmol / kg body weight while for X-ray applications Doses of 0.05 to 2.0 mmol / kg are generally effective, about an X-ray attenuation too to reach. Preferred dosages for most X-ray applications are 0.1 to 1.2 mmol of the lanthanum or heavy metal compound / kg Body weight.

Where The reporter is a radionuclide, dosages are from 0.01 to 100 mCi, preferably 0.1 to 50 mCi normally per 70 kg body weight suffice. Where the reporter is a superparamagnetic particle, the dosage is normally between 0.5 to 30 mg Fe / kg body weight be.

The Compounds of the present invention can be treated with conventional pharmaceutical or veterinary Excipients, for example emulsifiers, fatty acid esters, Gelling agents, stabilizers, antioxidants, osmolality adjusting Agents, buffers, pH adjusting agents, etc., and can be used in in a form suitable for parenteral administration, for example, injection or infusion or the administration is suitable directly into the vasculature. Therefore, the Compounds of the present invention in conventional pharmaceutical Administration forms, such as solutions, Suspensions and dispersions in physiologically acceptable carrier media, for example, water for injections.

The compounds according to the invention may therefore be suitable for administration using phy siologically acceptable carrier or excipient may be formulated in a manner that is fully within the skill of the art. For example, the compounds, optionally with the addition of pharmaceutically acceptable excipients, may be suspended or dissolved in an aqueous medium, the resulting solution or suspension then being sterilized.

parenterally administrable forms, e.g. intravenous solutions, should be sterile and free from physiologically unacceptable agents, and should a low osmolality to contribute to irritation or other adverse effects to minimize administration, and therefore the contrast medium should preferably isotonic or slightly hypertonic. Suitable vehicles include watery Vehicles, usually for the Administration of parenteral solutions such as sodium chloride injection, Ringer's injection, dextrose injection, Dextrose and sodium chloride injection, Lactated Ringer's injection and other solutions, as in Remington's Pharmaceutical Sciences, 15th Edition, Easton: Mack Publishing Co., pp. 1405-1412 and 1461-1487 (1975) and The National Formulary XIV, 14th Edition, Washington: American Pharmaceutical Association (1975). The solutions can Preservatives, antimicrobials, buffers and antioxidants, the usual for parenteral solutions used, excipients and other additives that contain compatible with the chelates and not with the production, Storage or use of products.

The The present invention will now be further illustrated by means of following examples. Unless otherwise indicated, all percentages are in weight percent specified.

example 1

Contrast agent for MR imaging of angiogenesis

Connection 1

Lysine (0.1 g, 0.7 mmol) becomes a solution of N ² - [3S-hydroxy-4- (N-hydroxyamino) 2R-isobutylsuccinyl] -L- (4-oxymethylcarboxy) phenylalanine-N ¹ -methylamide (prepared according to WO94 / 02447 , 0.3 g, 0.7 mmol) and DCC (N, N-dicyclohexylcarbodiimide) in dry DMF (N, N-dimethylformamide). The reaction mixture is stirred at ambient temperature, followed by TLC.

The Dispersion is over Night at + 4 ° C leave. The dispersion is filtered and the solvent becomes evaporated on a rotary evaporator before the substance by chromatography is cleaned.

Connection 2

Diethylentriaminpentaessigsäuredieanhydrid (17.9 g, 50 mmol) is dissolved in dry DMF and compound 1 (0.3 g, 0.5 mmol) in dry DMF is added. The reaction mixture is at increased Temperature stirred under nitrogen atmosphere. The reaction is followed by a TLC. The solvent is evaporated on a rotary evaporator and the substance by chromatography cleaned.

Gd (III) chelate of compound 2

To a solution of Compound 2 (0.4 g, 0.4 mmol) in water is added gadolinium oxide Gd ₂ O ₃ (0.1 g, 0.2 mmol) and the mixture is heated at 95 ° C. After filtration, the solution is evaporated and dried in vacuo at 50 ° C.

Example 2

Contrast agent for MR imaging of angiogenesis

Connection 3

Lysine (0.1 g, 0.7 mmol) becomes a solution of N- (4-octylphenyl) -3- (2-carboxyethyl) -6,7-dihydro-5H-thiazolo [3,2-a] pyrimidine -2-carboxamide (prepared according to EP-A-618 208 , 0.3 g, 0.7 mmol) and DDC (N, N'-dicyclohexylcarbodiimide) in dry DMF (N, N-dimethylformamide). The reaction mixture is stirred at ambient temperature, followed by TLC. The dispersion is left overnight at + 4 ° C. The dispersion is filtered and the solvent is evaporated on a rotary evaporator before the substance is purified by chromatography.

Connection 4

diethylenetriaminepentaacetic (17.9 g, 50 mmol) is dissolved in dry DMF and compound 3 (0.3 g, 0.5 mmol) in dry DMF is added. The reaction mixture is at increased Temperature stirred under nitrogen atmosphere. The reaction is followed by TLC. The solvent is evaporated on a rotary evaporator and the substance is passed through Purified by chromatography.

GD (III) chelate of compound 4

To a solution of compound 4 (0.4 g, 0.4 mmol) in water is added gadolinium oxide Gd ₂ O ₃ (0.1 g, 0.2 mmol) and the mixture is heated at 95 ° C. After filtration, the solution is evaporated and dried in vacuo at 50 ° C.

Example 3

contrast agents for nuclear medicine for the detection of angiogenesis

^99m Tc-chelate of compound 2

Compound 2 of Example 1 (1 mg) is dissolved in 0.1 N NaOH. SnCl ₂ · 2H ₂ O (100 ug) dissolved in 0.05 N HCl and a solution of 10-100 mCi ^99m Tc in the form of sodium pertechnetate in saline is added. The pH of the solution is adjusted to pH 7-8 by adding 0.5 M phosphate buffer (pH 5) in less than one minute. The reaction is followed by TLC and the substance is purified by chromatography.

Example 4

Contrast agent for nuclear medicine for detection of angiogenesis

^99m Tc-chelate compound 4

Compound 4 of Example 2 (1 mg) is dissolved in 0.1 N NaOH. SnCl ₂ · 2H ₂ O (100 ug) dissolved in 0.05 N HCl and a solution of 10-100 mCi ^99m Tc in the form of sodium pertechnetate in saline is added. The pH of the solution is adjusted to pH 7-8 by adding 0.5 M phosphate buffer (pH 5) in less than one minute. The reaction is followed by TLC and the substance is purified by chromatography.

Example 5

Contrast agent for nuclear medicine for detection of angiogenesis

An aqueous solution of ¹³¹ I ₂ (2 equivalents) and sodium perchlorate (1 equivalent) to an aqueous solution of N ² - [3S-Hydroxy-4-hydroxyamino) -2R-isobutylsuccinyl] -L-phenylalanine-N ¹ -methylamide ( manufactured according to WO94 / 02446 , 1 equivalent). The solvent is evaporated on a rotary evaporator and the substance is purified by chromatography.

Example 6

Preparation of DTPA-monoamide-gadolinium complex comprising a vector for targeting a VEGF receptor for MR detection of angiogenesis

a) Synthesis of 6,7-dimethoxy-3H-quinazolin-4-one

A mixture of 2-amino-4,5-dimethoxybenzoic acid (9.9 mg, 0.050 mmol) and formamide (5 mL) was heated at 190 ° C for 6 hours. The mixture was cooled to 80 ° C and poured onto water (25 ml). Precipitated material was filtered off, washed with water and dried in vacuo. yield 1.54 g (15%), brown powder. The structure was confirmed by ¹ H (500 MHz) and ¹³ C NMR (125 MHz) analysis.

b) Synthesis of 4-chloro-6,7-dimethoxyquinazoline

A suspension of the compound of a) (1.03 g, 5.00 mmol) in phosphorus oxychloride (20 ml) was refluxed for 3 hours. The dark solution was concentrated and the residue was taken up in ethyl acetate. The organic phase was washed with saturated sodium bicarbonate solution and dried (MgSO ₄ ). The solution was filtered through a short silica column and concentrated to give 392 mg (35%) of a cream colored material. ¹ H NMR (300 MHz) and ¹³ C NMR (75 MHz) spectra were in agreement with the structure.

c) Synthesis of [4- (6,7-dimethoxyquinazolin-4-ylamino) -phenyl] acetic acid

A mixture of the compound of b) (112 mg, 0.500 mmol) and 4-aminophenylacetic acid (76 mg, 0.50 mmol) in 2-propanol (8 ml) was refluxed for 3 hours. The reaction mixture was cooled and precipitated material was isolated, washed with 2-propanol and dried in vacuo. Yield 183 mg (97%), pale yellow solid material. The structure was verified by ¹ H-NMR (500 MHz) and ¹³ C-NMR (125 MHz) analysis. Further characterization was performed using MALDI mass spectrometry (α-cyano-4-hydroxycinnamic acid matrix), giving m / z for [MH] ⁺ at 341, expecting 340.

d) Synthesis of t-butyl (6- {2- [4- (6,7-dimethoxyquinazolin-4-ylamino) phenyl] acetylamino} hexyl) carbamate

To a suspension of the compound of c) (38 mg, 0.10 mmol) and N-Boc-1,6-diaminohexane hydrochloride (25 mg, 0.10 mmol) in DMF (2.0 mL) was N, N-diisopropylethylamine (34 mL, 0.20 mmol). To the clear solution was added N- (3-dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (19 mg, 0.10 mmol) and 1-hydroxybenzotriazole (15 mg, 0.10 mmol). The reaction mixture was stirred at room temperature overnight and then poured into 25 ml of water containing sodium carbonate (2.5 g) and sodium chloride (4.0 g). Organic material was extracted into chloroform and the organic phase was washed with water and dried (Na ₂ SO ₄ ). The solution was filtered and concentrated. The product was purified by column chromatography (silica, chloroform / methanol / acetic acid 85: 10: 5) and finally lyophilized from acetic acid. Yield 54 mg (90%), yellow-white solid material (acetate). The product was characterized by MALDI mass spectrometry (α-cyano-4-hydroxycinnamic acid matrix), giving m / z for [MH] ⁺ at 539 as expected.

Further characterization was performed using ¹ H (500 MHz) and ¹³ C (125 MHz) NMR spectroscopy.

e) Synthesis of N- (6-aminohexyl) - [4- (6,7-dimethoxyquinazolin-4-ylamino) phenyl] acetamide hydrochloride

The compound of d) (27 mg, 0.050 mmol) was dissolved in dioxane (3 mL) by gentle warming. To the solution was added 4N HCl in dioxane (0.5 ml). The reaction mixture was stirred overnight and concentrated in vacuo to give a quantitative yield of the title compound. Characterization was carried out using MALDI mass spectrometry (α-cyano-4-hydroxycinnamic acid matrix), giving m / z for [MH] ⁺ at 439 as expected. Further characterization was performed using analytical HPLC (Vydac 218TP54 column, gradient 12 to 24% B over 20 minutes, A = water / 0.1% TFA, B = acetonitrile / 0.1% TFA, flow rate 1.0 ml / min), giving a single product peak with a retention time of 13.0 minutes detected at 340 nm. Characterization was also carried out by NMR spectroscopy, giving ¹ H (500 MHz) and ¹³ C (125 MHz) spectra in accordance with the structure.

f) Synthesis of a DTPA monoamide derivative for gadolinium chelation (Structure shown above)

N, N-Diisopropylethylamine (17 μl, 0.10 mmol) was added to a suspension of the compound of e) (0.05 mmol) and DTPA anhydride (179 mg, 0.500 mmol) in DMF (5 ml). The mixture was stirred at room temperature for 2 hours and concentrated in vacuo. HPLC analysis (Vydac 218TP54 column, gradient 16-28% B over 20 minutes, A = water / 0.1% TFA, B = acetonitrile / 0.1% TFA, flow rate 1.0 mL / min) gave a product peak 7.9 min, which was shown by LC-MS (ESI) to correspond to the title compound (m / z for [MH] ⁺ at 813, expected 814). The product was purified by preparative HPLC (Vydac 218TP1022 column, gradient 16-28% B over 60 minutes, A = water / 0.1% TFA, B = acetonitrile / 0.1%. TFA, flow rate 10.0 ml / min, detection at 254 nm), giving a yield of 6.7 mg of purified material. Analysis of the purified material by analytical HPLC showed a shift in retention time to 5.6 min (analytical conditions as described above), which was shown by MALDI mass spectrometry to correspond to the iron complex, giving m / z at 870 for the complex and 816 for the free ligand.

g) Preparation of the Gadolinium Complex the compound of f)

The Compound of f) (0.1 mg) was dissolved in an aqueous solution of gadolinium trichloride (Conc. 2 mg / ml, 0.1 ml). The mixture was over Night stirred. The quantitative conversion to the gadolinium complex was determined by MALDI mass spectrometry (Α-cyano-4-hydroxycinnamic acid matrix) verified what m / z peaks at 970, 992 and 1014 for the gadolinium complex (Gadolinium, gadolinium / sodium or gadolinium / disodium) and at 816/838 corresponding to the free ligand / sodium complex. None Trace of the iron complex could be detected.

Example 7

Preparation of a DTPA-bisamide gadolinium complex comprising a vector for targeting a VEGF receptor for MR detection of angiogenesis

a) Synthesis of a DTPA bisamide derivative for gadolinium chelation (Structure shown above)

Analytical HPLC of the reaction mixture in Example 6f) also gave a peak at 16.8 min, which was shown by LC-MS (EIS) analysis to be equal to the DTPA-bisamide shown above, m / z at 1233 for [MH] ⁺ as expected and yielded 616.6 as expected for [MH ₂ ] ²⁺ . The product was purified by preparative HPLC (conditions as described in Example 6f) to give 14 mg of the pure material after lyophilization. Analysis of the purified material by analytical HPLC showed a shift in retention time from 16.8 minutes (in the crude mixture) to 11.1 minutes due to formation of the iron complex during purification, as verified by MALDI mass spectrometry (α-cyano). 4-hydroxycinnamic acid matrix), giving m / z at 1291 for the iron complex and 1237 for the free ligand.

b) Preparation of the Gadolinium Complex the compound of a)

The Compound of a) was treated with gadolinium trichloride as in Example 6g). After 2 hours reaction time showed MALDI mass spectrometry a conversion to the gadolinium complex, which is m / z at 1391 for the gadolinium complex and 1235 for revealed the free ligand.

Example 8

Carboxymethyl- [2- (carboxymethyl {2- [carboxymethyl - ({2- [3 - ({3-oxo-2- [2- (pyridin-2-ylamino) -ethyl] -2,3-dihydro-1 H -isoindole-5-carbonyl} amino) propionylamino] ethylcarbamoyl} methyl) amino] ethyl} amino) ethyl] amino} acetic acid (12)

a) 4-methylisophthalic acid (2)

To a THF (130 ml) solution of 3-bromo-4-methylbenzoic acid (5.0 g, 23.25 mmol) under nitrogen and cooled to -78 ° C (dry ice / methanol) MeMgBr in ether (3.0 M, 8.5 mL, 25.57 mmol) in such Given speed that the temperature did not exceed -75 ° C. They left the Temperature then to -60 ° C increase and after the evolution of gas had ceased, the solution became again cooled to -78 ° C. BuLi in hexane (1.6M, 29.06mL, 46.50mmol) was then added dropwise admitted that the temperature did not rise above -75 ° C. The mixture was then stirred at this temperature for 15 minutes before crushed dry ice (4.4 g, 100 mmol) was added. The precipitate was stirred vigorously while stirring the temperature was allowed to rise freely to ambient temperature. The mixture was acidified using 6N HCl and the solid material was collected by filtration, washed with diethyl ether and dried. Recrystallization from water gave the cream-colored pure compound (81%) mp 296-298 ° C (sublimed). NMR confirms the expected structure.

b) 4-methylisophthalmethyl ester (4)

A Mixture of compound (2) (2.83 g, 15.71 mmol), thionyl chloride (50 ml) and DMF (3 drops) was refluxed for 2 hours heated. After cooling to room temperature was excess thionyl chloride removed under reduced pressure (rotary evaporator). The dark oil that received was dissolved in carbon tetrachloride (30 ml) Pyridine (1 ml, 12.43 mmol) and methanol (20 ml) and treated at Stirred ambient temperature for 2 hours. The solvents were evaporated and the residue was purified by flash chromatography: silica, hexane / EtOAc (9: 1).

c) 4-Bromethylisophthalsäuredimethylester (5)

A Mixture of (4) (0.96 g, 4.61 mmol), dibenzoyl peroxide (56 mg, 0.23 mmol) and N-bromosuccinimide (NBS) (0.82 g, 4.61 mmol) in carbon tetrachloride (20 ml) was heated at reflux for 20 hours. After cooling up Room temperature and filtration, the solvent was evaporated to a yellow oil to surrender. Flash chromatography: silica, hexane / EtOAc (7: 3) the pure connection.

d) Methyl 3-oxo-2- [2- (pyridin-2-ylamino) ethyl] 2,3-dihydro-1H-isoindole-5-carboxylate (6)

A solution of (5) (511 mg, 1.78 mmol) in toluene (10 mL) was treated with Et ₃ N (744 μL, 5.33 mmol) and N, 1-pyridin-2-yl-ethane-1,2 -diamine (244 mg, 1.78 mmol) [prepared by treating 2-bromopyridine with excess ethylenediamine and pyridine] and refluxing the mixture for 6 hours. After cooling to room temperature and evaporation of the solvent, the residue was purified by flash chromatography: silica, CH ₂ Cl ₂ / acetone (3: 2).

e) 3-Oxo-2- [2-pyridin-2-ylamino) -ethyl] 2,3-dihydro-1H-isoindole-5-carboxylic acid (7)

A methanol solution (6 ml) of (6) (301 mg, 0.97 mmol) and 1 N NaOH (3 ml) was stirred at ambient temperature for 24 hours. The solution was acidified using 1 M NaHSO ₄ solution and the precipitated product was collected by filtration, washed thoroughly with water and dried over P ₂ O ₅ / Bluegel for 24 hours.

f) 3 - ({3-Oxo-2- [2- (pyridin-2-ylamino) ethyl] 2,3-dihydro-1H-isoindole-5-carbonyl} amino) -propionic acid tert-butyl ester (8th)

A solution of (7) (166 mg, 0.56 mmol), N-methylmorfolin (185 μl, 1.68 mmol), BOP (322 mg, 0.73 mmol) and H-β-ala-OtBu (152 mg , 0.84 mmol) in DMF (5 ml) was stirred at ambient temperature for 20 hours. The mixture was diluted with ethyl acetate (10 ml) and then washed once each with H ₂ O, NaHCO ₃ , 10% KHSO ₄ and brine (5 ml), dried (MgSO ₄ ) and concentrated. Flash chromatography (silica, EtOAc) gave the ester (8) as a white solid.

g) 3 - ({3-Oxo-2- [2- (pyridin-2-ylamino) ethyl] 2,3-dihydro-1H-isoindole-5-carbonyl} amino) -propionic acid (9)

A solution of ester (8) (252 mg, 0.60 mmol), TFA (4 mL) and CH ₂ Cl ₂ (8 mL) was stirred at ambient temperature for 3 hours. The mixture was evaporated to dryness and the residue was purified by flash chromatography (silica, EtOH / NH ₄ OH 19: 1) to give (9) as a cream foam. NMR confirms the structure.

h) {2- [3 - ({3-Oxo-2- [2- (pyridin-2-ylamino) ethyl] -2,3-dihydro-1H-isoindole-5-carbonylamino) -propionylaminol-ethyl} - carbamic acid tert-butyl ester (10)

To a solution of the acid (9) (20 mg, 0.054 mmol) in DMF (2 mL) was N-methylmorfolin (NMM) (16.40 mg, 0.162 mmol), BOP (Castro's reagent) 31.05 mg, 0.070 mmol) and the BOC-protected diamine (13 mg, 0.081 mmol) and the mixture was stirred at ambient temperature for 20 hours. After dilution with EtOAc (5 ml), the solution was washed once with H ₂ O, sat. NaHCO ₃ , 10% KHSO ₄ and brine. The organic phase was dried (MgSO ₄ ) and concentrated. Flash chromatography (silica, 1: 1 CH ₂ Cl ₂ / acetone). MALDI-MS; 510.59.

i) 3-Oxo-2- [2- (pyridin-2-ylamino) -ethyl] -2,3-dihydro-1H-isoindole-5-carboxylic acid [2- (2-aminoethylcarbamoyl) -ethyl] -amide (11)

A CH ₂ Cl ₂ solution (3 mL) of (10) (20 mg, 0.039 mmol) and TFA (2 mL) was stirred under ambient conditions for 4 hours. The reaction mixture was concentrated and the residue was purified by flash chromatography (silica, 19: 1, CH ₂ Cl ₂ / acetone) to give (4) as a white solid. MALDI-MS; 410.48.

j) Carboxymethyl [2- (carboxymethyl) {2- [carboxymethyl-f {2- [3 - [{3-oxo-2- [2- [pyridin-2-ylamino) ethyl] -2,3-dihydro -1H-isoindole-5-carbonyl} -amino) -propionylaminolethylcarbamoyl} -methyl) -amino] -ethyl} -amino) -ethyl] -amino} -acetic acid (12)

A solution of (11) (15 mg, 0.36 mmol), N, N-diisopropylamine (17 μl, 0.10 mmol) and DTPA anhydride (129 mg "0.36 mmol) in DMF (2 ml) at ambient temperature for 3 hours touched and concentrated in vacuo. The residue was by preparative HPLC (acetonitrile / 0.1% TFA in water).

Example 9

Preparation of a DTPA monoamide gadolinium complex comprising a vector for targeting a bFGF receptor for MR detection of angiogenesis

a) Synthesis of tert-butyl {3- [2- (4-chlorobenzyloxy) -2- (2,4-dichlorophenyl) ethyl] -3H-imidazol-1-yl} acetic acid

1- [2- (4-chlorobenzyloxy) -2- (2,4-dichlorophenyl) -ethyl] -1H-imidazole (1g, 2.25mmol) and tert -butyl bromoacetate (1mL, 6.8mmol) dissolved in 15 ml of acetonitrile and over Night under reflux heated. TLC showed a full conversion of the starting material. The solvent was cooled, evaporated in vacuo and the residual oil was dissolved in chloroform and triturated with ether. The product was analyzed by MALDI mass spectrometry identified and in the next Step used without further purification.

b) Synthesis of: {3- [2- (4-Chlorobenzyloxy) -2- (2,4-dichlorophenyl) ethyl] -3H-imidazol-1-yl} acetic acid

The Compound of a) (500 mg, 1 mmol) was dissolved in 2 ml of dichloromethane and dissolved in cooled in an ice bath. 2 ml of trifluoroacetic acid was added, the ice bath was removed and the reaction mixture was stirred for 1 hour. TLC showed a full conversion of the starting material. The solvent was removed in vacuo and the product in the next step without further Cleaning used.

c) Synthesis of: [6- (2- {3- [2- (4-Chlorobenzyloxy) -2- (2,4-dichlorophenyl) ethyl] -3 H -imidazol-1-yl} -acetylamino) -hexyl] -carbamic acid tert-butyl ester

The Product of b) became c) by that described for Example 6d) Procedure was converted, and the product was purified by flash chromatography cleaned.

d) Synthesis of: N- (6-aminohexyl) -2- {3- [2- (4-chlorobenzyloxy) -2- (2,4-dichlorophenyl) ethyl] -3H-imidazol-1-yl} -acetamide

The Product of c) became d) by that described for example 6e) Procedure converted. The product was used without further purification.

e) Synthesis of: ((2 - {[2- (Bis-carboxymethylamino) -ethyl] -carboxymethylamino} ethyl) - {[6- (2- {3- [2- (4-chlorobenzyloxy) -2- (2 , 4-dichlorophenyl) -ethyl] -3H-imidazol-1-yl} acetylamino) -hexylcarbamoyl] -methyl} -amino) -acetic acid

• f) Die Herstellung des Gadoliniumkomplexes der Verbindung e) wurde durch die in Beispiel 6g) beschriebene Prozedur ausgeführt.

The product of d) was converted to e) by the procedure described for example 6f). Purification was carried out by preparative HPLC as described in Example 6f).

• f) The preparation of the gadolinium complex of compound e) was carried out by the procedure described in Example 6g).

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Claims (10)

A method of producing an image of a living human or non-human animal subject to which a contrast agent has previously been administered, comprising generating an image of at least a portion of the subject to which the contrast agent has been delivered, characterized in that the contrast agent is a composition of a material of the formula I is VLR (I) wherein V is a vector moiety having an affinity for an angiogenesis-related endothelial cell receptor, L is a linker moiety or a bond, and R is a detectable moiety, characterized in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species that provides a variety of markers that are detectable in in vivo imaging. A method for monitoring the effect of treating a human or non-human animal subject with a drug to combat or provoke angiogenesis-related effects, the method comprising detecting the uptake of a previously administered formula I composition VLR (I) wherein V is a vector moiety having an affinity for an angiogenesis-related endothelial cell receptor, L is a linker moiety or a bond, and R is a detectable moiety, characterized in that V is a non-peptidic organic group, or V is peptidic and R is a macromolecular or particulate species that provides a variety of markers detectable in in vivo imaging, angiogenesis-related endothelial cell receptors, by producing an image of at least a portion of the subject, with administration and detection optionally, but preferred repeatedly, eg before, during or after treatment with the drug. A method according to claim 1 or claim 2, wherein V is a vector for is an integrin receptor. A method according to claim 1 or claim 2, wherein V is a vector for is a fibronectin receptor. A method according to claim 1 or claim 2, wherein V is a vector for the VEGF receptor is. A method according to claim 1 or claim 2, wherein V is a vector for is the urokinase plasminogen activator receptor (UPAR). The method of claim 1 or claim 2 wherein V is selected from the list of vector units comprising: N ² - [3S-hydroxy-4- (N-hydroxyamino) 2R-isobutylsuccinyl] -L- (4-oxymethylcarboxy) phenylalanine N ¹ -methylamide, N- (4-octylphenyl) -3- (2-carboxyethyl) -6,7-dihydro-5H-thiazolo [3,2-a] pyrimidine-2-carboxamide, N ² - [3S-hydroxy 4-hydroxyamino) -2R-isobutylsuccinyl] -L-phenylalanine N ¹ -methylamide, N- (6-aminohexyl) - [4- (6,7-dimethoxy-quinazolin-4-ylamino) -phenyl] -acetamide hydrochloride, 3- Oxo-2- [2- (pyridin-2-ylamino) -ethyl] -2,3-dihydro-1H-isoindole-5-carboxylic acid [2- (2-aminoethylcarbamoyl) -ethyl] -amide, and N- (6 amino-hexyl) -2- {3- [2- (4-chloro-benzyloxy) -2- (2,4-dichlorophenyl) -ethyl] -3H-imidazol-1-yl} acetamide. A method according to claim 1 or claim 2 wherein VLR is selected from the following residues: Gd (III), ^99m Tc or ¹³¹ I ₂ chelates of the DTPA derivatives of vector units of claim 7. A method according to any one of claims 1 to 8, wherein R is a radionuclide is. The method of claim 9, wherein R is an iodine or Metal Radionuclide is.